Functional assay of high-density lipoprotein

ABSTRACT

This invention provides novel assays that are prognostic and/or diagnostic for atherosclerosis or risk of atherosclerosis. It was discovered that high density lipoprotein (HDL) or components thereof can prevent the oxidation of lipids (e.g., lipids present in LDLs) and can also repair (reduce) already oxidized lipids and thereby reduce the inflammatory response associated with and characteristic of atherosclerotic plaque formation. Moreover it was a discovery of this invention that individuals vary in the ability of their HDL to afford such protection. Thus an assay of HDL protective and/or repair activity provides a highly effective assay for risk of atherosclerosis and its associated pathologies and such assays are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. Ser. No. 10/455,243,now U.S. Pat. No. 6,869,568, filed on Jun. 5, 2003, which is aDivisional of U.S. Ser. No. 09/541,468, now U.S. Pat. No. 6,596,544,filed Mar. 31, 2000, all of which are incorporated herein by referencein their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant No: HL30568,awarded by the National Institutes of Health. The Government of theUnited States of America may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the diagnosis of atherosclerosis. Inparticular this invention provides improved assays

BACKGROUND OF THE INVENTION

Cardiovascular disease is a leading cause of morbidity and mortality,particularly in the United States and in Western European countries.Several causative factors are implicated in the development ofcardiovascular disease including hereditary predisposition to thedisease, gender, lifestyle factors such as smoking and diet, age,hypertension, and hyperlipidemia, including hypercholesterolemia.Several of these factors, particularly hyperlipidemia andhypercholesteremia (high blood cholesterol concentrations) provide asignificant risk factor associated with atherosclerosis.

Cholesterol is present in the blood as free and esterified cholesterolwithin lipoprotein particles, commonly known as chylomicrons, very lowdensity lipoproteins (VLDLs), low density lipoproteins (LDLs), and highdensity lipoproteins (HDLs). Concentration of total cholesterol in theblood is influenced by (1) absorption of cholesterol from the digestivetract, (2) synthesis of cholesterol from dietary constituents such ascarbohydrates, proteins, fats and ethanol, and (3) removal ofcholesterol from blood by tissues, especially the liver, and subsequentconversion of the cholesterol to bile acids, steroid hormones, andbiliary cholesterol.

Maintenance of blood cholesterol concentrations is influenced by bothgenetic and environmental factors. Genetic factors include concentrationof rate-limiting enzymes in cholesterol biosynthesis, concentration ofreceptors for low density lipoproteins in the liver, concentration ofrate-limiting enzymes for conversion of cholesterols bile acids, ratesof synthesis and secretion of lipoproteins and gender of person.Environmental factors influencing the hemostasis of blood cholesterolconcentration in humans include dietary composition, incidence ofsmoking, physical activity, and use of a variety of pharmaceuticalagents. Dietary variables include amount and type of fat (saturated andpolyunsaturated fatty acids), amount of cholesterol, amount and type offiber, and perhaps amounts of vitamins such as vitamin C and D andminerals such as calcium.

As indicated above, high blood cholesterol concentration is one of themajor risk factors for vascular disease and coronary heart disease inhumans. Elevated low density lipoprotein cholesterol (“LDL-cholesterol”)and total cholesterol are directly related to an increased risk ofcoronary heart disease (see, e.g., Anderson et al. (1987) JAMA,257:2176-2180).

Although high levels of total cholesterol and LDL-cholesterol are riskfactors in developing atherosclerosis and vascular diseases, adeficiency of high density lipoprotein cholesterol (hereafter“HDL-cholesterol”) has recently been recognized as a risk factor fordeveloping these conditions. Several clinical trials support aprotective role of HDL-cholesterol against atherosclerosis. A study hasshown that for every 1-mg/dl increase in HDL-cholesterol in the blood,the risk for coronary vascular disease is decreased by 3% in women (see,Gordon et al. (1989) Circulation, 79: 8-15).

It is widely believed that HDL is a “protective” lipoprotein (Vega andGrundy (1996) Curr. Opin. Lipidology, 7, 209-216) and that increasingplasma levels of HDL may offer a direct protection against thedevelopment of atherosclerosis. Numerous studies have demonstrated thatboth the risk of coronary heart disease (CHD) in humans and the severityof experimental atherosclerosis in animals are inversely correlated withserum HDL cholesterol (HDL-C) concentrations (Russ et al. (1951) Am. J.Med., 11: 480-493; Gofman et al. (1966) Circulation, 34: 679-697; Millerand Miller (1975) Lancet, 1: 16-19; Gordon et al. (1989) Circulation,79: 8-15; Stampfer et al. (1991) N. Engl. J. Med., 325: 373-381; Badimonet al. (1989) Lab. Invest., 60: 455-461).

While HDU LDL ratios have appear to provide a good marker for risk ofatherosclerosis and heart disease on a population level, HDL and/or LDLmeasurements have proven to be poor prognostic indicators at anindividual level. In particular individuals with high HDL:LDL ratioshave been observed with severe atherosclerosis, while conversely,individuals with very low HDL:LDL ratios have been identified with noevidence of atherosclerosis.

SUMMARY OF THE INVENTION

This invention provides novel assays that are prognostic and/ordiagnostic for atherosclerosis or risk of atherosclerosis. The assaysare based, in part, on elucidation of a mechanism by which HDL affordsprotection against plaque formation. In particular, it was a discoveryof this invention that HDL or components can prevent the oxidation oflipids (e.g., lipids present in LDLs) and can also repair (reduce)already oxidized lipids and thereby reduce the inflammatory responseassociated with and characteristic of atherosclerotic plaque formation.Moreover it was a discovery of this invention that individuals vary inthe ability of their HDL to afford such protection. Thus an assay of HDLprotective and/or repair activity provides a highly effective assay forrisk of atherosclerosis and its associated pathologies.

Thus, in certain embodiments, this invention provides methods ofevaluating the risk for atherosclerosis in a mammal by evaluating theability of the animal's HDL to repair (reduce) oxidized phospholipids.The methods can involve providing a biological sample from the mammalwhere the sample comprises a high-density lipoprotein (HDL) or acomponent thereof (e.g., apo A-I, paraoxonase, platelet activatingfactor acetylhydrolase, etc.), contacting the high-density lipoproteinwith an oxidized phospholipid; and measuring a change in the amount ofoxidized or non-oxidized phospholipid where the reduction or absence ofchange in the amount of oxidized phospholipid indicates the mammal is atrisk for atherosclerosis. In certain embodiments, the oxidizedphospholipids is provided in combination with low density lipoprotein(LDL).

Suitable oxidized phospholipids include an oxidized phospholipid thatcauses a monocytic reaction. Certain preferred phospholipids include,but are not limited to the oxidized form of lipids selected from thegroup consisting of1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC),1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC),1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC),1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEIPE). In one particularly preferred embodiment, the oxidized phospholipidis a component of (present in) a low density lipoprotein.

The oxidized phospholipid (or reduced phospholipid) can be determined byany convenient method. Such methods include, but are not limited to massspectrometry, liquid chromatography, thin layer chromatography,fluorimetry, radioisotope detection, antibody detection, and detecting asignal from a label that indicates an oxidized phospholipid. Fluorescentlabels (e.g., 2′,7′-dichlorodihydrofluorescine diacetate, rhodamine,cis-parinaric acid, NBD, cis-parinaric acid cholesteryl ester,diphenylhexatriene propionic acid), and dichlorofluorescein diacetate(DCFH-DA), are particularly preferred.

In certain embodiments, the detecting comprises a chromatography methodselected from the group consisting of fast performance liquidchromatography (FPLC).

Preferred samples include fluid or tissue samples containing HDL.Particularly preferred samples include, but are not limited to wholeblood or blood fractions (e.g., serum, plasma, etc.).

The sample can be used directly, or alternatively, HDL may be isolatedfrom the sample. Thus, for example, in certain embodiments, magneticbead reagents are used to remove non-HDL cholesterol. The change and/oramount of oxidized phospholipid can be determined relative to knownlevels for the subject population and/or by reference to variouscontrols. Such controls include, but are not limited to the change inamount of oxidized phospholipid produced by contacting the oxidizedphospholipid with HDL known to reduce levels of oxidized phospholipid,the change in amount of oxidized phospholipid produced by contacting theoxidized phospholipid with HDL known to be deficient in the ability toreduce levels of oxidized phospholipid, and the change in phospholipidproduced in the same experiment run without HDL or with HDL present at alower concentration.

The mammal can be a human or a non-human. Particularly preferred mammalsinclude, but are not limited to humans, non-human primates, canines,felines, murines, bovines, equines, porcines, and lagomorphs. The humanmay be a human diagnosed as having a low HDL:LDL ratio and/or as beingat risk for atherosclerosis.

In another embodiment this invention provides methods of evaluating therisk for atherosclerosis in a mammal by measuring the ability of themammal's HDL to protect lipids from oxidation. The methods preferablyinvolve providing a biological sample from the mammal where the samplecomprises a high-density lipoprotein (HDL), contacting the high densitylipoprotein with a phospholipid, subjecting the phospholipid tooxidizing conditions; and measuring a change in the amount of oxidizedor non-oxidized phospholipid where a change in the amount of oxidized ornon-oxidized phospholipid indicates the mammal is at risk foratherosclerosis. In a preferred embodiment the phospholipid is providedin a low density lipoprotein (LDL). Particularly preferred phospholipidsare phospholipids that, when oxidized, cause a monocytic reaction. Suchphospholipids include, but are not limited to1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC),1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC),1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC),1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEIPE).

In certain embodiments the phospholipid is subjected to oxidizingconditions by contacting the phospholipid with an oxidizing agent, e.g.,an agent selected from the group consisting of hydrogen peroxide,13(S)-HPODE, 15(S)-HIPETE, HPODE, HPETE, HODE, and HETE. The detectionof oxidized or reduced phospholipid can be by any convenient method,with the methods described herein (e.g., described above) being mostpreferred. Particularly preferred detection labels include but are notlimited to 2′,7′-dichlorodihydrofluorescine diacetate, rhodamine,cis-parinaric acid, NBD, cis-parimic acid cholesteryl ester, anddiphenylhexatisene propionic acid. Preferred samples are as describeabove and herein. In the case of blood or blood fraction samples, themethod may involve direct use of the blood or blood fraction orisolation of HDL from the blood or blood fraction.

The change and/or amount of oxidized phospholipid can be determinedrelative to known levels for the subject population and/or by referenceto various controls. Such controls include, but are not limited to thechange in amount of oxidized phospholipid produced by contacting theoxidized phospholipid with HDL known to reduce levels of oxidizedphospholipid, the change in amount of oxidized phospholipid produced bycontacting the oxidized phospholipid with HDL known to be deficient inthe ability to reduce levels of oxidized phospholipid, and the change inphospholipid produced in the same experiment run without HDL or with HDLpresent at a lower concentration.

Preferred mammals assayed according to the methods of this inventioninclude humans and non humans, e.g., as described above. Particularlypreferred subjects are humans diagnosed as having a low HDL:LDL ratioand/or as being at risk for atherosclerosis.

In still another embodiment this invention provides kits for evaluatingthe risk for atherosclerosis in a mammal. The kits preferably comprise acontainer containing one or more oxidized or non-oxidized phospholipids,and instructional materials providing protocols for the assays describedherein. The kits optionally include a label for detecting oxidizedphospholipid and/or optionally, an oxidizing agent (e.g., 13(S)-HPODE,15(S)-HPETE, HPODE, HPETE, HODE, and HETE). In certain embodiments, thekit comprises a container containing one or one or more oxidizedphospholipids, and the instructional materials describe assaying HDL forthe ability to reduce oxidized lipids. In other embodiments, the kitcomprises a container containing one or more non-oxidized (reduced)phospholipids, and the instructional materials describe assaying HDL forthe ability to protect lipids (e.g., lipids in LDL) from oxidation.

In still another embodiment, this invention provides test devices forthe assays of this invention. The test device preferably comprises aninert porous substrate having a receiving area, the porous substratebeing juxtaposed to a transport medium, the transport medium beingjuxtaposed to a test membrane comprising a reagent for detecting anoxidized lipid. The test device optionally includes a non-oxidized lipidand an oxidizing agent or an oxidized phospholipid.

Definitions

The terms “low density lipoprotein” or “LDL” are defined in accordancewith common usage of those of skill in the art. Generally, LDL refers tothe lipid-protein complex which when isolated by ultracentrifugation isfound in the density range d=1.019 to d=1.063.

The terms “high density lipoprotein” or “HDL” are defined in accordancewith common usage of those of skill in the art. Generally “HD” refers tolipid-protein complex which when isolated by ultracentrifugation isfound in the density range of d=1.063 to d=1.21.

The term “Group I HDL” refers to a high density lipoprotein orcomponents thereof (e.g., apo A-I, paraoxonase, platelet activatingfactor acetylhydrolase, etc.) that reduce oxidized lipids (e.g., in lowdensity lipoproteins) or that protect oxidized lipids from oxidation byoxidizing agents.

The term “Group II HDL” refers to an HDL that offers reduced activity orno activity in protecting lipids from oxidation or in repairing (e.g.,reducing) oxidized lipids.

The term “HDL component” refers to a component (e.g., molecules) thatcomprises a high density lipoprotein (HDL). Assays for HDL that protectlipids from oxidation or that repair (e.g., reduce oxidized lipids) alsoinclude assays for components of HDL (e.g., apo A-I, paraoxonase,platelet activating factor acetylhydrolase, etc.) that display suchactivity.

A “monocytic reaction” as used herein refers to monocyte activitycharacteristic of the “inflammatory response” associated withatherosclerotic plaque formation. The monocytic reaction ischaracterized by monocyte adhesion to cells of the vascular wall (e.g.,cells of the vascular endothelium), and/or chemotaxis into thesubendothelial space, and/or differentiation of monocytes intomacrophages.

The term “absence of change” when referring to the amount of oxidizedphospholipid refers to the lack of a detectable change, more preferablythe lack of a statistically significant change (e.g., at least at the85%, preferably at least at the 90%, more preferably at least at the95%, and most preferably at least at the 98% or 99% confidence level).The absence of a detectable change (e.g., when scoring a positive resultfor Group I HDL) can also refer to assays in which oxidized cholesterollevel changes, but not as much as in the absence of the HDL or withreference to other positive or negative controls.

The following abbreviations may be used herein: PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine;PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phsophocholine;ChC18:2: cholesteryl linoleate; ChC18:2—OOH: cholesteryl linoleatehydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine;PON: paraoxonase; HPF: Standardized high power field; PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phsophocholine;PON: paraoxonase; HPF: Standardized high power field; BL/6: C57BL/6J;C3H:C3H/HeJ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one embodiment of a test for assaying activity of HDLin protecting lipids from oxidation.

FIG. 2 illustrates the removal of seeding molecules from LDL by apo A-I.Butylated hydroxy toluene (BHT) was added to freshly isolated plasma toa concentration of 20 μM and was fractionated by gel filtrationchromatography using an FPLC system with two Superose 6 columnsconnected in series and eluting with normal saline. The fractionscontaining LDL were pooled. Purified apo A-I (100 μg/ml) was added tothe LDL (1 mg/ml) and incubated for 2 hrs at 37° C. with gentle mixingin normal saline. The LDL and apo A-I were then re-isolated by FPLC orby centrifugation. Re-isolated LDL is designated “LDL after A-I” and there-isolated apo A-I is designated “A-I after LDL”.

FIG. 3, panels A, B, and C, illustrate the resistance of LDL tooxidation following incubation with apo A-I. LDL was rapidly isolated byFPLC from seven normal human donors and 1 mg/ml LDL incubated with 100μg/ml apo A-I followed by re-isolation of the LDL and apo A-I asdescribed in FIG. 2. Cocultures of artery wall cells were incubated withsham-treated LDL (LDL sham) or with IDL that was incubated with apo A-Iand was re-isolated (LDL after A-I), or with sham-treated apo A-I (A-Isham). To other coculture wells was added reconstituted LDL that wasprepared by incubating “LDL after A-I” plus the lipids extracted from“A-I after LDL” (A-I lipids after LDL+LDL after A-I). The cocultureswere incubated for 8 hrs at 37° C. in the presence of 10% LPDS. Thesupernatants were collected and analyzed for lipid hydroperoxide levels.Monocyte adhesion was determined on one set of the cocultures and theothers were washed and incubated with culture medium without serum orLPDS for 16 hrs. This conditioned medium was collected and analyzed formonocyte chemotactic activity. Panel A demonstrates the lipidhydroperoxide levels of supernatants. Panel B demonstrates monocyteadherence and Panel C contains the values for monocyte chemotacticactivity. The figure is a representative of seven separate experimentsusing LDL from 7 different normal donors and cocultures and monocytesfrom different donors. The values are mean±SD of quadruplicatecocultures. The asterisks indicate p<0.0004.

FIG. 4, panels A and B, illustrate the effect of pretreatment of LDLwith apo A-I peptide mimetics on LDL oxidation and chemotactic activity.Freshly isolated LDL was incubated at 250 μg/ml with buffer (Sham LDL),with the apo A-I mimetic peptide 37 pA at 100 μg/ml or with the controlpeptide 40 P at 100 μg/ml. The incubation was conducted in M199 for 2hrs at 37° C. with gentle mixing. LDL and the peptides were subsequentlyre-isolated as in FIG. 1. Cocultures of artery wall cells were incubatedwith sham-treated LDL (Sham LDL), or LDL that was incubated with the apoA-I mimetic peptide (LDL after 37 pA), or with the control peptide (LDLafter 40 P), sham-treated 37 pA (37 pA sham), or sham-treated 40 P (40 Psham). To other coculture wells was added reconstituted LDL that wasprepared by incubating “LDL after 37 pA” plus the lipids extracted from“37 pA after LDL” (37 pA lipids after LDL+LDL after 37 pA). Theseadditions were incubated with human artery wall cocultures for 8 hrs inthe presence of 10% LPDS. The supernatants were collected and analyzedfor lipid hydroperoxide levels (Panel A). The cocultures were thenwashed and were incubated with culture medium without serum or LPDS for8 hrs. The conditioned medium was then collected and analyzed formonocyte chemotactic activity (Panel B). The data indicate mean±SD ofvalues obtained from quadruplicate cocultures in three separateexperiments. Asterisks indicate p<0.0014.

FIG. 5, panels A, B, C, and D, show the bioactivity of lipids extractedby apo A-I-Freshly isolated LDL (1 mg/ml) was incubated with apo A-I(100 μg/ml) and re-isolated as indicated in FIG. 1. Lipids wereextracted from “A-I after LDL” by chloroform-methanol extraction andseparated with solid phase extraction chromatography as described inMethods. The fatty acid (FA) or neutral lipid (NL) fractions wereevaporated to dryness and were incubated with 200 μl of M199 containing10% LPDS at 37° C. for 5 minutes with intermittent gentle vortexing.Fatty acids (FA-A-I after LDL, Panel A and Panel B), or neutral lipids(NL-A-I after LDL, Panel C and Panel D), were then incubated at theindicated quantities with either 100 μg PAPC or 250 μg “LDL after A-I”in a total volume of 1 ml of M199 containing 10% LPDS at 37° C. for 3hrs. This treated PAPC or LDL in M 199 containing 10% LPDS was thenincubated with HAEC at 37° C. for 4 hrs. The supernatants were removedand assayed for lipid hydroperoxide content (Panel A and Panel C) asdescribed herein. The cells were washed and monocyte adhesion wasdetermined (Panel B and Panel D) as described herein.

FIG. 6, panels A through H, illustrate the removal of 13-HPODE and15-HPETE by apo A-I from LDL. Freshly isolated LDL (1 mg/ml) wasincubated alone (LDL sham), or with apo A-I (100 μg/ml) in M199 for 2hrs, with gentle mixing. For controls, 100 μg/ml apo A-I was incubatedalone in M 199 for 2 hrs (A-I Sham, Panel A and Panel E) or 1 mg/mlfreshly isolated LDL was incubated alone in M 199 for 2 hrs (LDL Sham,Panel B and Panel F) with gentle mixing at 37° C. The LDL and apo A-Iwere then re-isolated by centrifugation using Millipore molecular weightcut-off filters (100 kDa). Lipids were extracted from apo A-I and fromLDL and were analyzed by reverse phase HPLC. Panel C and Panel Gdemonstrate the decrease in the 13-HPODE and 15-HPETE peaks in LDLfollowing incubation with apo A-I (LDL after A-I) and FIG. 6D and FIG.6H demonstrate the increase in 13-HPODE and 15-HPETE respectively in thelipid extract from apo A-I after incubation with and separation from LDL(A-I after LDL).

FIG. 7, panels A and B, illustrate seeding molecules in LDL from C57BL/6and C3H/HeJ mouse strains on a chow diet. LDL was isolated from plasmaobtained from groups (n=5 each group) of the lesion susceptible C57BL/6(BL/6) and from the lesion resistant C3H/HeJ (C3H) mice. The LDL wasincubated (at 100 μg/ml) with human apo A-I (at 100 μg/ml) with gentlemixing at 37° C. and then re-isolated by FPLC as indicated in FIG. 2.Reconstitution of LDL with lipids removed by apo A-I was carried out asdescribed in FIG. 3 and incubated with aortic wall cell cocultures. Theabbreviations are the same as in FIG. 3. Panel A shows data on lipidhydroperoxides formed and Panel B demonstrates the chemotactic activitythat was induced. The values shown are mean±SD of quadruplicatecocultures. The asterisks indicate p<0.0015.

FIG. 8, panels A and B, show that injection of apoA-I (but not apoA-II)into mice renders the mouse LDL resistant to oxidation by human arterywall cells. Groups of C57BL/6 mice (n=5) were injected in the tail veinwith 100 μg per animal of apo-AI, apoA-II or with saline alone. Bloodsamples were removed at time points, LDL was isolated and incubated withcocultures for 8 hrs. Culture supernatants were assayed for lipidhydroperoxides (Panel A) and for monocyte chemotactic activity (Panel B)as described in Methods. The figure depicts the mean±SD of quadruplicatesamples from a representative experiment. The asterisks indicatep<0.0001 as compared to 0 time. Identical results were obtained in twoout of two separate experiments.

FIG. 9, panels A and B, show that infusion of human apo A-I into humansrenders their LDL resistant to oxidation by human artery wall cells. Sixindividuals (described herein) were infused with human apoA-I/phosphatidylcholine discs at 40 mg apo A-I/kg body weight during a4-hr period. Plasma was prepared 2 hrs before and 6 hrs following thestart of the infusion (i.e., 2 hrs after completion of the infusion).LDL was isolated by FPLC and incubated (at 100 μg/ml) with coculturesfor 8 hrs. Culture supernatants were collected and subjected to lipidextraction and were assayed for hydroperoxide content (Panel A). Thecocultures were washed and incubated in culture medium without serum orLPDS for 8 hrs and the conditioned medium was analyzed for monocytechemotactic activity (Panel B). Mean±SD of quadruplicate cocultures arepresented and asterisk indicates p<0.0173 for panel A; p<0.0077 forpanel B.

FIG. 10, panels A and B, show that HDL or HDL associated enzymes renderLDL resistant to oxidation by human artery wall cells. Freshly isolatedLDL was incubated at 250 μg/ml with buffer (Sham treated LDL), with HDLat 350 μg/ml (HDL treated LDL) or with purified PON at 1×10⁻² U/ml (PONtreated LDL). The incubation was conducted in M199 for 3 hrs at 37° C.with gentle mixing. LDL was subsequently re-isolated by centrifugationusing Millipore molecular weight cut-off filters (100 kDa) and wasincubated with human artery wall cocultures for 8 hrs in the presence of10% LPDS. The supernatants were removed and analyzed for lipidhydroperoxides (Panel A); the cocultures were washed and incubated withculture medium without serum or LPDS. After 8 hrs the medium wascollected and analyzed for monocyte chemotactic activity (Panel B). Thedata indicate mean±SD of values obtained from quadruplicate coculturesin three separate experiments. Asterisks indicate significance at thelevel of p<0.0008.

FIG. 11, panels A and B, show that Apo A-I removes substances from humanartery wall cells and renders the cells unable to oxidize LDL.Cocultures were incubated with 50 μg/ml of apo A-I or apo A-II or weresham treated for 8 hrs. The conditioned media containing either apo A-Ior apo A-II were removed and in some cases transferred to othercocultures that had been treated identically and served as targetcocultures. LDL was added at 250 μg/ml to the target cocultures that hadbeen sham treated (Cultures sham treated), or to target cocultures thathad been treated with apo A-I which had been removed (Cultures afterA-I), or treated with apo A-II which had been removed (Cultures afterA-II). LDL was also added at 250 μg/ml to target cocultures that hadbeen treated with apo A-I or apo A-II and to which was added theconditioned media containing either apo A-I or apo A-II from the firstset of cocultures (Cultures after A-I+A-I after cultures), (Culturesafter A-II+AII after cultures), respectively. The target cocultures wereincubated for 8 hrs in M199 containing 10% LPDS and LDL with or withoutthe additions (conditioned media) from the first set of cocultures. Somecocultures received 250 μg/ml of LDL plus 50 μg/ml of apo A-I at thestart of the 8 hr incubation and this was continued for a total of 16hrs (Co-incubated A-I). The supernatants were removed and assayed forlipid hydroperoxides (Panel A) and the cocultures were washed and freshM199 without serum or LPDS was added and incubated for 8 hrs and assayedfor monocyte chemotactic activity (Figure Panel B). Values are mean±SDfrom three separate experiments utilizing LDL from different donors.Asterisks indicate significance at the level of p<0.001.

FIG. 12, panels A and B, show that an apo A-I peptide mimetic removessubstances from human artery wall cells and renders the cells unable tooxidize LDL. Human aortic wall cocultures were incubated with mediumalone (Sham treated), with an apo A-I mimetic peptide at 100 μg/ml (37pA treated) or with control peptide at 100 μg/ml (40P treated) for 8hrs. The cocultures were then washed and freshly isolated LDL was addedand incubated in M199 containing 10% LPDS for an additional 8 hrs. Themedia were removed and assayed for lipid hydroperoxides (Panel A). Thecocultures were then washed and were incubated with culture mediumwithout serum or LPDS for an additional 8 hrs and assayed for monocytechemotactic activity (Panel B). The data represent mean±SD of valuesobtained from quadruplicate cocultures in three separate experiments.Asterisks indicate significance at the level of p=0.011.

FIG. 13, panels A and B, show that HDL and its associated enzyme PONrender human artery wall cells unable to oxidize LDL. Human aortic wallcocultures were incubated with medium alone (Sham treated), with HDL at350 μg/ml (HDL treated), or with purified paraoxonase at 1×10×⁻² U/ml(PON treated) for 8 hrs. The cocultures were then washed and freshlyisolated LDL was added at 250 μg/ml and incubated in M199 containing 10%LPDS for an additional 8 hrs. The media were collected and analyzed forlipid hydroperoxides (Panel A). The cocultures were then washed and wereincubated with culture medium without serum or LPDS for 8 hrs and thesupernatant was collected and analyzed for monocyte chemotactic activity(Panel B). The data represent mean±SD of values obtained fromquadruplicate cocultures in three separate experiments. Asterisksindicate significance at the level of p<0.011.

FIG. 14, panels A, B, and C, show that pretreatment of human artery wallcells with linoleic acid results in increased levels of lipidhydroperoxides, monocyte chemotactic activity and removal of 13-HPODE byapoA-I. Two sets of cocultures were incubated for 18 hours at 37° C.with 100 μM oleic acid (C18:1), or linoleic acid (C18:2) in M199 with10% LPDS. The medium was removed and the cultures washed three times.Fresh medium without fatty acids was added and the cultures wereincubated at 37° C. for an additional 3 hrs. IDL at 250 μg/ml was thenadded to one set of the cocultures in M199 containing 10% LPDS andincubated for 8 hrs. The medium was then removed and lipidhydroperoxides (Panel A) and monocyte chemotactic activity (Panel B)determined. To the second set of cocultures (Panel C) apo A-I was addedat 100 μg/ml and incubated for three more hours with gentle mixing at37° C. The supernatant was removed, apo A-I was separated by FPLC, andthe hydroperoxide content of the lipid extract of the supernatants thatdid not receive apo A-I (Culture supernatant) and the lipid extract fromapo A-I (Apo A-I lipid extract) were determined as described in Methodswhich are expressed as ng per well. Values are mean±SD of triplicatedeterminations. The asterisks denote p<0.001.

FIG. 15, panels A, B, and C, show that 13(S)-HPODE accelerates theformation of bioactive oxidized phospholipids from PAPC. Ten μg of PAPCwith 1.0 μg of 13(S)-HPODE (stippled bars) or with vehicle alone (openbars) were mixed and evaporated forming a thin film and allowed tooxidize in air for the times shown. Following extraction withchloroform-methanol, the samples were analyzed by ESI-MS in the positiveion mode. The data represent the levels of1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine (POVPC, m/z 594,panel A), 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC, m/z610, panel B), and 1-palmitoyl-2-(5,6-epoxyisoprostaneE₂)-sn-glycero-3-phosphocholine (PEIPC, m/z 828, panel C) relative to aninternal standard (0.1 μg DMPC) that was added with the PAPC. The valuesare the mean±SD of triplicate samples. The asterisks indicate p<0.01.13(S)-HPODE alone did not give a signal for m/z 594, 610 or 828 (datanot shown).

FIG. 16, panels A, B, and C, show that 13(S)-HPODE, 15(S) HPETE or H₂O₂accelerate in a dose dependant manner the formation of oxidizedphospholipids from PAPC. Ten μg of PAPC was mixed with the indicatedmicrograms of 13(S)-HPODE (Panel A), or 15(S)-HPETE (Panel B) andevaporated forming a thin film and allowed to oxidize in air for 8 hrs.In Panel C, ten μg of PAPC was evaporated forming a thin film and H₂O₂was added at the indicated concentrations and allowed to oxidize for 8hrs. Following extraction with chloroform-methanol, the samples wereanalyzed by ESI-MS in the positive ion mode. The data represent thelevels of PAPC, m/z 782; POVPC, m/z 594; PGPC, m/z 610; and PEIPC, m/z828 relative to an internal standard (0.1 μg DMPC) that was added withthe PAPC. The values are the mean±SD of triplicate samples. 13(S)-HPODEalone, 15(S)-HPETE alone or H₂O₂ did not give a signal for m/z 594, 610or 828 (data not shown). Asterisks indicate significant differences atp<0.001.

FIG. 17, panels A, B, and C, show that 13(S)-BPODE stimulates thenon-enzymatic formation of cholesteryl linoleate hydroperoxide(Ch18:2-OOH). 13(S)-BPODE 0.5 μg/ml (Panel A) or cholesteryl linoleate10 μg/ml (Panel B) or cholesteryl linoleate 10 μg/ml together with 13(S)HPODE 0.5 μg/ml (Panel C) in chloroform/methanol (2:1, v/v) containing0.01% BHT was briefly swirled to mix and evaporated to dryness underargon and allowed to undergo air oxidation in a laminar flow hood for 6hrs. The lipids were solubilized in 50 μl of chloroform and analyzed forthe presence of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) byRP-HPLC as described herein.

FIG. 18 shows that purified paraoxonase destroys the bioactivity of theoxidized phospholipids. Oxidized PAPC, (Ox-PAPC), POVPC (m/z 594), PGPC(m/z 610) or PEIPC (m/z 828) were incubated in test tubes in M199without, or with 1×10⁻² U/ml purified human paraoxonase (+PON) for 3 hrswith gentle mixing at 37° C. Paraoxonase was removed from the mixtureand the lipids were incubated with human aortic wall cocultures in M199with 10% LPDS for 8 hrs at 37° C. The cocultures were then washed andincubated with fresh media without serum or LPDS for an additional 8 hrsat 37° C. The supernatants were removed and analyzed for monocytechemotactic activity. Data are mean±SD for quadruplicate cocultures.Asterisks indicate significant differences at the level p<0.0001.

FIG. 19, panels A, B, C, and D, show that HDL from patients withangiographically documented coronary atherosclerosis despite normalIHDL-cholesterol levels that is deficient in paraoxonase activity, doesnot protect LDL from oxidation by artery wall cells, and does notdestroy the biologic activity of oxidized phospholipids. These patientshad angiographically documented coronary atherosclerosis, despite normaltotal cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterollevels. The patients were not diabetic nor on hypolipidemic medications.Paraoxonase activity was determined as described in Methods for 24patients and 29 age and sex matched normal subjects (Panel A). Data from14 previously reported patients and from 19 previously reported normalsubjects are included in panel A together with data from an additional10 patients and age and sex matched normal subjects. The ability of HDLfrom the additional 10 patients and controls to protect a control LDLagainst oxidation by artery wall cells is shown in Panel B as determinedby lipid hydroperoxide formation as described herein and in Panel C bymonocyte chemotactic activity which was determined as described herein.The data in panel C includes data previously reported for 5 patients and4 normal subjects together with data from the additional 10 patients andtheir age and sex matched normal subjects. The data in Panel D representa new approach, namely the ability of patient and normal HDL (n=10 foreach group) to inhibit the biologic activity of oxidized PAPC (Ox-PAPC).In each instance 100 μg/ml of Ox-PAPC was incubated with 250 μg/ml ofHDL in test tubes in 10% LPDS in M199 at 37° C. with gentle mixing for 4hrs. The HDL-Ox-PAPC mixture was then added to endothelial monolayersand monocyte binding determined. Data are the mean±SD of quadruplicatecocultures and the asterisk indicates a significant difference at thelevel of p<0.01 for Panel A; p<0.001 for LDL vs LDL+patient HDL,p<0.0001 for LDL+cont. HDL vs LDL+patient HDL in Panel B; p<0.009 forcontrol LDL vs LDL+Control HDL, p<0.000008 for LDL+Control HDL vsLDL+Patient HDL in Panel C; p<0.009 for Ox-PAPC vs Ox-PAPC +Patient HDL,p<0.0001 for Ox-PAPC +Control HDL vs Ox-PAPC+Patient HDL in Panel D.

FIG. 20 illustrates a three step model for LDL oxidation by artery wallcells: Step1—LDL is seeded. Step 2—LDL is trapped in the artery wall andreceives further seeding molecules. Step 3—When a critical level ofseeding molecules relative to phospholipids is reached in the LDL, anon-enzymatic oxidation process generates POVPC, PGPC, and PEIPC. LDLthat is formed from the hydrolysis of VLDL in the circulation maycontain “seeding molecules”. Alternatively, LDL may enter thesubendothelial space (A), where it is seeded with reactive oxygenspecies (ROS) delivered from the artery wall cells (STEP 1). While thecartoon depicts this as occurring in the subendothelial space, STEP 1might actually occur in the microcirculation. If the LDL is seeded inthe subendothelial space it might remain there becoming trapped in theextracellular matrix (B) or the seeded LDL could exit into thecirculation (C) and re-enter the subendothelial space at another sitewhere it would become trapped in the extracellular matrix (D). In STEP 2the artery wall cells generate and transfer additional or different ROSto the trapped seeded LDL. This transfer could occur within the cell, atthe cell surface, or in an adjacent protected microdomain. Followingthis transfer of reactive oxygen species to the seeded and trapped LDL,a non-enzymatic propagation of lipid oxidation occurs (STEP 3). Thisresults in the formation of specific oxidized phospholipids that induceNF-κB activation, monocyte binding, MCP-1 production, and M-CSFproduction and which are present in mildly oxidized LDL (minimallymodified LDL; MM-LDL). As indicated, normal HDL is capable of blockingeach and every step in the formation of MM-LDL.

FIG. 21. D-4F or scrambled D-4F (Sc D-4F) at 100 μg/mL was added to thedrinking water of apoE null mice (n=3 for each group) overnight. In themorning the mice were bled and their HDL isolated using Magnetic BeadReagent (Polymedco) and 5 μg of mouse HDL-cholesterol was tested in thecell-free assay. The results clearly show improvement in HDL functionafter administration of D-4F.

FIG. 22. The data in the FIG. 21 were normalized so that thefluorescence from LDL-Ox-PAPC alone was set at a value of 1.0. The othersamples were then normalized to this value. The results show how thesedata can be used to construct an HDL-inflammatory index. Values lessthan 1.0 represent anti-inflammatory HDL; values>1.0 representpro-inflammatory HDL.

FIG. 23. Three subjects with anti-inflammatory HDL (subjects 1, 2, and3) were compared to three subjects with pro-inflammatory HDL (subjectsA, B, and C) in the cell-free assay. Five micrograms of HDL-cholesterolisolated using Magnetic Bead Reagent (Polymedco) from each subject wasadded to the LDL+Ox-PAPC in this assay.

FIG. 24. HDL was isolated using Magnetic Bead Reagent (Polymedco) fromserum or plasma from a single normal individual and tested in thecell-free assay. Five micrograms of HDL-cholesterol was added to thecell-free assay after being isolated from either serum or plasma. Theresults were indistinguishable with HDL isolated from either serum orplasma.

DETAILED DESCRIPTION

This invention provides novel assays that are prognostic and/ordiagnostic for atherosclerosis or risk of atherosclerosis. The assaysare based, in part, on elucidation of a mechanism by which HDL affordsprotection against plaque formation.

It has been noted that freshly isolated low density lipoprotein (LDL)contains lipid hydroperoxides (Sevanian et al. (1997) J. Lipid Res.,38:419-428). We believe that LDL oxidation requires that the LDL be“seeded” with reactive species before it can be oxidized. The presenceof oxidized lipids results in an “inflammatory response; the inductionof monocyte binding, chemotaxis, and differentiation into macrophages.This process underlies plaque formation characteristic ofatherosclerosis.

More particularly, without being bound to a theory, it is believed thatthe biologically active lipids in mildly oxidized LDL (m/z 594, 610, and828) are formed in a series of three steps. The first step is theseeding of LDL with products of the metabolism of linoleic andarachidonic acid as well as with cholesteryl hydroperoxides. The secondstep involves trapping of LDL in the subendothelial space and thedelivery to this trapped LDL of additional reactive oxygen speciesderived from nearby artery wall cells. The third step is thenon-enzymatic oxidation of LDL phospholipids that occurs when a criticalthreshold of “seeding molecules” (e.g., 13-hydroperoxyoctadecadienoicacid [13(S)-HPODE] and 15-hydroperoxyeicosatetrenoic acid [15(S)-HPETE]) is reached in the LDL. This results in the formation of specificoxidized lipids (m/z 594,610,828) that induce monocyte binding,chemotaxis, and differentiation into macrophages. We present evidencewhich indicates that when the “seeding molecules” reach a criticallevel, they are approximately two orders of magnitude more potent thanhydrogen peroxide in causing the non-enzymatic oxidation of a major LDLphospholipid, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine(PAPC) resulting in the formation of the three biologically activeoxidized phospholipids (m/z 594, 610, and 828) (Watson et al. (1997) JBiol Chem 272:13597-13607; Watson et al. (1999) J Biol Chem274:24787-24798).

The experiments described herein also indicate that, in contrast to thecase for normal HDL, BDL taken from a relatively rare subset ofpatients, those with angiographically documented coronary artery diseasewho had perfectly normal levels of LDL-cholesterol, HDL-cholesterol, andtriglycerides and who were not diabetic and who were not takinghypolipidemic medications did not protect LDL against oxidation by humanartery wall cells and failed to inhibit the biologic activity ofoxidized PAPC.

Thus, we have identified two sets of subjects: 1) Those subjects whoseHDL affords protection against the formation of oxidized lipids and/orreduces or eliminates these oxidized lipids and hence protects againstthe associated inflammatory processes of atherosclerosis (designatedherein as Group I HDLs); and 2) Those subjects whose HDL does not affordprotection against the formation of oxidized lipids, and/or does notreduce or eliminate these oxidized lipids, particularly oxidized LDL(designated herein as Group II HDLs). It is believed that thedifferences in the HDL activity between these two sets of subjectsaccounts, at least in part, for the lack of predictability offered byconventional HDL assays. It is also believed that subjects in thissecond subset are at considerably greater risk for atherosclerosis andits associated complications. An assay that distinguishes between thesetwo sets of subjects (i.e., between subjects having Group I HDLs andsubjects having Group II HDLs) is of significant prophylactic anddiagnostic value.

As a prophylactic assay, the methods of this invention allowidentification of individuals of particularly high risk foratherosclerosis. Upon such identification, such subjects can adopt morefrequent testing, dietary adjustments, monitoring and regulation ofblood pressure, and the like. As a diagnostic assay, the methods of thisinvention supplement traditional testing methods (e.g., HDL:LDL ratios,etc.) to identify subjects known to be at risk who may prove resistantto conventional therapeutic regimens and alter the prescribed treatment.Thus, for example, where a subject is diagnosed with early stages ofatherosclerosis, a positive test using the assays of this invention mayindicate additional drug intervention rather than simplydietary/lifestyle changes.

In one embodiment the assays exploit the discovery that the “Group IHDLs” can actually reduce and/or eliminate oxidized phospholipids. Thus,Group I HDLs can be identified by providing a biological sample fromsaid mammal (e.g., serum) where the biological sample comprises ahigh-density lipoprotein (HDL), contacting the high-density lipoproteinwith an oxidized phospholipid and/or oxidized phospholipids(s) combinedwith a low density lipoprotein (LDL); and measuring a change in theamount of oxidized or non-oxidized phospholipid wherein a reduction inthe amount of oxidized phospholipid indicates the mammal has Group IHDLs and, hence, is at lower risk for atherosclerosis. Conversely, whereno significant change in oxidized phospholipid is observed, the subjecthas Group II HDLs and is at increased risk for atherosclerosis.

In another embodiment, the assays of this invention exploit thediscovery that Group I HDLs can prevent the oxidization of LDLs and/orphospholipid-containing components of LDLs. These assays preferablyinvolve providing a biological sample from a mammal where the samplecomprises a high-density lipoprotein (HDL), contacting the high densitylipoprotein with a phospholipid (e.g., isolated phospholipid and/or witha low density lipoprotein), subjecting the phospholipid to oxidizingconditions; and measuring a change in the amount of oxidized ornon-oxidized phospholipid. A change in the amount of oxidized ornon-oxidized phospholipid indicates that the HDL is a Group II HDL andis not protecting the lipid from oxidation. Thus subject mammal is thusat increased risk for atherosclerosis. Where there is reduced, or nosubstantial change in oxidized or unoxidized phospholipid, the HDLaffords protection against lipid oxidation and the subject is at reducedrisk for atherosclerosis and associated pathologies.

The assays of this invention are rapid, simple, inexpensive, and canreadily be formatted as a “home test kit”.

I. HDL Activity Assays.

As indicated above, in certain preferred embodiments, the assays of thisinvention can be performed in several formats. In one format, the HDL isassayed for the ability to reduce the level of oxidized phospholipid(e.g., in a low density lipoprotein). In another format, the HDL isassayed for the ability to protect a phospholipid from oxidation by anoxidizing agent.

Both assay formats require provision of a biological sample containingHDL (or components thereof) contacting the HDL (or KDL components) witha lipid (oxidized or not depending on the assay), and detecting theamount of oxidized lipid or lipid that is not oxidized. The assaysdiffer in that the first assay contacts the HDL (or HDL component) of anoxidized lipid (or LDL comprising such lipid), while the second assaycontacts the HDL to a lipid that is not oxidized, and contacting thelipid with an oxidizing agent to evaluate the protection from oxidationafforded by the HDL.

A) Providing a Biological Sample Comprising HDL.

In certain preferred embodiments the assays are performed using abiological sample from the organism/subject of interest. While theassays are of great use in humans, they are not so limited. It isbelieved similar HDL subtypes exist essentially in all mammals and thusthe assays of this invention are contemplated for veterinaryapplications as well. Thus, suitable subjects include, but are notlimited to humans, non-human primates, canines, equines, felines,porcines, ungulates, largomorphs, and the like.

A suitable biological sample includes a sample of any biologicalmaterial (e.g., fluid, cell, tissue, organ, etc.) comprising highdensity lipoproteins (HDLs) or components thereof. One particularlypreferred tissue is liver tissue. In a most preferred embodiment, thebiological sample is a blood sample. As used herein a blood sampleincludes a sample of whole blood or a blood fraction (e.g., serum,plasma, etc.). The sample can be fresh blood or stored blood (e.g., in ablood bank) or blood fractions. The sample can be a blood sampleexpressly obtained for the assays of this invention or a blood sampleobtained for another purpose which can be subsampled for the assays ofthis invention.

The sample may be pretreated as necessary by dilution in an appropriatebuffer solution, heparinized, concentrated if desired, or fractionatedby any number of methods including but not limited toultracentrifugation, fractionation by fast performance liquidchromatography (FPLC), or precipitation of apolipoprotein B containingproteins with dextran sulfate or other methods. Any of a number ofstandard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

In certain embodiments, the sample is treated to remove non-HDLcholesterol. This can be accomplished by any of a number of methods knowto those of skill in the art. In one embodiment, this is accomplished bythe use of a commercially supplied magnetic bead reagent, e.g., asdescribed in the examples.

B) Contacting the HDL With a Lipid.

HDL from the biological sample is then contacted with a lipid (oxidizedor not depending on the assay as described above) or collection oflipids (isolated lipid(s) or presented as an LDL). The HDL can be fullyisolated, partially isolated, or the whole (e.g., unfractionated)biological sample can be contacted with the lipid. Methods of partiallyor completely isolating HDL are known to those of skill in the art (see,e.g., Havel, et al. (1955) J Clin Invest 43:1345-1353; Navab et al.(1997) J Clin Invest 99:2005-2019; Carroll and Rudel (1983) J Lipid Res24:200-207, McNamara et al. (1994) Clin Chem 40:233-239, Grauholt et al.(1986) Scandinavian J Clin Lab Invest 46:715-721; Warnick et al. (1982)Clin Chem 28:1379-1388; Talameh et al., (1986) Clin Chimica Acta158:33-41).

In a preferred embodiment, the lipid that is contacted comprises one ormore lipids (preferably phospholipids) capable of being oxidized.Preferred lipid(s) include, but are not limited to reduced (notoxidized) 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine,1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC),1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC),1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC),1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE),1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE),1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEIPE).

These lipids are illustrative and not intended to be limiting. Othersuitable lipids can be readily identified by those of ordinary skill inthe art. This is accomplished simply by contacting the lipid(s) inquestion with an oxidizing agent (e.g., hydrogen peroxide, HPODE, HPETE,HODE, HETE, etc.) and measuring the amount of oxidized lipid produced.Alternatively, the “oxidized lipid/LDL can be assayed for its ability toinduce a response characteristic of atherosclerotic plaque formation(e.g., induction of monocyte adhesion and/or chemotaxis, and/ordifferentiation in a culture of vascular endothelial cells).

The lipid(s) can be presented as “isolated” or “partially isolated”lipid(s) or may be presented/contacted in the form of a low densitylipoprotein (LDL). The LDL can be an LDL isolated from an organism or asynthetically assembled/created LDL. Means of isolating or synthesizinglipids (e.g., phospholipids), and/or LDLs are well known to those ofskill in the art (see, e.g., Havel, et al. (1955) J Clin Invest43:1345-1353; Navab et al. (1997) J Clin Invest 99:2005-2019; Carrolland Rudel (1983) J Lipid Res 24:200-207, etc.).

C) Detecting the Level of Oxidized Lipid.

As indicated above, the assays can involve detecting the amount ofoxidized lipid, or conversely lipid that is not oxidized. Since, incertain embodiments, the lipid content of the assay is essentiallyconstant, a measurement of oxidized lipid or change in oxidized lipidprovides a measure of lipid that is not oxidized or a change in theamount of lipid that is not oxidized and vice versa.

Methods of measuring oxidized lipids are well known to those of skill inthe art (see, e.g., Vigo-Pelfrey et al. Membrane Lipid Oxidation, VolumeI-III. CRC Press). such methods include, but are not limited to massspectrometry, absorption spectrometry (e.g., using UV absorbance at 234nm), liquid chromatography, thin layer chromatography, and the use ofvarious “oxidation-state” sensitive reagents (e.g., dichlorofluoresceindiacetate (DCFH-DA), etc.), e.g., in various redox reactions.

Previously known methods for measuring oxidized lipids (e.g., lipidperoxides), include the Wheeler method, iron thiocyanate method,thiobarbituric acid method, and others. The Wheeler method (Wheeler(1932) Oil and Soap, 9: 89-97) is that in which oxidized lipid isreacted with potassium iodide to isolate iodine, which is then titratedwith a sodium thiosulfate standard solution. In the iron thiocyanatemethod (Stine et al. (1954) J. Dairy Sci.,37: 202) oxidized lipidperoxide is mixed with ammonium thiocyanate and ferrous chloride, andthe blue color from the resulting iron thiocyanate is calorimetricallydetermined. In the thiobarbituric acid method (Tappel and Zalkin (1959)Arch. Biochem. Biophys.,80: 326) the lipid peroxide is heated underacidic conditions and the resulting malondialdehyde is condensed withthiobarbituric acid to form a red color dye, which is thencalorimetrically measured.

In another approach, it has been demonstrated that peroxidase decomposeslipid peroxides and that the resulting reaction system colors intenselywith increasing quantities of lipid peroxide, if an adequate hydrogendonor is present in the reaction system (see, e.g., U.S. Pat. No.4,367,285) Thus, in one embodiment, the assays of this invention mayutilize a peroxidase and a hydrogen donor.

Many peroxidases are suitable. In preferred embodiments, the peroxidaseemployed in the present invention is preferably any of the commerciallyavailable horseradish peroxidases.

In certain embodiments, the hydrogen donor employed in the presentinvention is any of the known oxidizable compounds which, preferably,generate color, fluorescence or luminescence upon oxidation. Theconventional coloring, fluorescent, luminescent reagents may beutilized. The known coloring reagents which may be employed include, butare not limited to guaiacol, 4-aminoantipyrine with phenol,4-aminoantipyrine with N,N-dimethylaniline, 3-methyl-2-benzothiazolinonewith dimethylaniline, ortho-dianisidine, and the like. Typically usefulfluorescent reagents include, but are not limited to homovanillic acid,p-hydroxyphenylacetic acid, and the like. Suitable luminescent reagentsinclude but are not limited to luminol and the like. All of thesereagents are mentioned merely for exemplification, and not forlimitation, of the hydrogen donor of the present invention.

The amount of the hydrogen donor employed is preferably at leastequimolar, preferably not less than two moles, per mole of lipidperoxide contained in test sample. The amount may be varied dependingupon the size of the sample and the content of the lipid peroxide in thesample.

Suitable reaction mediums which can be employed include, but are notlimited to dimethylglutarate-sodium hydroxide buffer solution, phosphatebuffer solution and, Tris-hydrochloric acid buffer solution is normallyfrom about pH 5 to about pH 9.

A typical (high volume) assay (3 ml) may contain a 50 mMdimethylglutarate-sodium hydroxide buffer solution (pH 6.0) containing0.03% (W/V) of 4-aminoantipyrine, 0.04% (V/V) of N,N-dimethylaniline and4.5 units of peroxidase. In a typical measurement, the assay solution ispreliminarily warmed to 37° C. and 50 μL of a test sample containinglipid peroxide is added. The mixture is incubated at 37° C. for 15minutes and, the intensity of the color generated is measured suing aspectrophotometer at a wavelength of, for example, 565 nm. The amount ofthe lipid peroxide in the sample is calculated from the extinctionvalue.

Such factors as the pH at the time of reaction, the reaction period, themeasuring wavelength, etc., may be varied depending upon the reagentsemployed. Suitable conditions can be selected according to thecircumstances.

Another class of assays for oxidized lipids is described in U.S. Pat.No. 4,900,680. In this approach, an oxidized lipid (e.g., ahydroperoxide) is reacted with a salt or hydroxide of a transition metalwhich produces a cation having a valency of 2, a complex of a transitionmetal having a valency of 2, a heme, a heme peptide, a heme protein, ora heme enzyme. The resultant active oxygen and oxygen radicals reactwith a luminescent substance, and light emitted by this reaction isoptically measured. Examples of a catalyst acting on a lipidhydroperoxide to produce active oxygen species such as active oxygen oroxygen radicals are: a transition metal salt which produces a cationhaving a valency of 2 (e.g., ferrous chloride, ferrous sulfate,potassium ferricyanide, each of which produces Fe²⁺; manganous chlorideor manganous sulfate, each of which produces Mn²⁺; or cobalt chloride orcobalt sulfate, each of which produces Co²⁺); a hydroxide of thetransition metals described above; a complex of a transition metalhaving a valency of 2 (e.g., Fe^(II)-porphyrin complex); a heme protein(e.g., cytochrome C, hemoglobin, or myoglobin); a heme peptide (e.g., acompound obtained by decomposing a heme protein by a protease such aschymotrypsin or trypsin); and a heme enzyme (e.g., horseradishperoxidase or prostaglandin peroxidase).

Preferred catalyst compounds include, but are not limited to, a hemeprotein, a heme peptide, or a heme enzyme. Most usually, the hemeprotein such as cytochrome C is used due to easy handling. Theconcentration of the catalyst compound preferably ranges from about 0.1μg/ml to about 1,000 μg/ml and usually falls within the range of about 1μg/ml to about 200 μg/ml. For example, best luminous efficiency can beobtained when the concentration is about 10 μg/ml for cytochrome C,about 120 μg/ml for cytochrome C heme peptide; and about 10 μg/ml forhorseradish peroxidase.

The luminescent substance is not limited to a specific one, provided itreacts with active oxygen or an oxygen radical to emit light. Examplesof such a compound include, but are not limited to polyhydroxyphenols(e.g., pyrogallol, perprogalline etc.), phthaladine derivatives (e.g.,luminol, isoluminol, etc.), indol derivatives (e.g., indoleacetic acid,skatole, tryptophan, etc.); thiazolidine derivatives (e.g., Cypridinacealuciferin, lophine, etc.), an acrydine derivatives (e.g., lucigenine),oxalic acid derivatives (e.g., bistrichlorophenyloxalate); and1,2-dioxa-4,5-azine derivatives. The concentration of the luminescentsubstance varies depending on the compound used. The concentration ispreferably 0.1 μg/ml or more. When luminol is used, its concentration ismost preferably 1 μg/ml.

Measurements are preferably performed in a weak basic solution of aluminescent reagent such as a heme protein and luminol. A preferred pHvalue ranges from about pH 9 to about pH 10. Many buffers are suitable.On preferred buffer is a borate buffer (H₃BO₃—KOH), a carbonate buffer(Na₂CO₃—NaHCO₃), a glycine buffer (NH₂CH₂ COOH—NaOH), or the like. Theborate buffer is most preferred.

In order to prevent oxygen dissolved in the luminescent reagent solutionfrom interfering analysis of a very small amount of oxidized lipid, theluminescent reagent solution is desirably purged with an inert gas toremove oxygen to obtain a stable measurement value. Examples of theinert gas are nitrogen gas and argon gas.

The concentration of the oxidized lipid in the sample is calculatedbased on a calibration curve. The calibration curve can be formedaccording to standard methods, e.g., by using a material selected frommethyl linolate hydroperoxide, arachidonic acid hydroperoxide,phosphatidylcholine hydroperoxide, phosphatidylethanolaminehydroperoxide, and triacylglycerol hydroperoxide.

In preferred embodiments, the assays of this invention utilizefluorescent materials whose fluorescence is altered by oxidation state.Such fluorescent materials are well known to those of skill in the artand include, but are not limited to 2′7′-dichlorodihydrofluorescinediacetate, rhodamine cis-parinaric acid, NBD, cis-parinimic acidcholesteryl esters, diphenylhexatriene proprionic acid, and the like.The use of such indicators is illustrated in the examples.

In certain embodiments, antibodies can be used to specifically detectoxidized phospholipids. Thus, for example U.S. Pat. No. 6,225,070provides a panel of monoclonal antibodies (“E0 antibodies”) that haveunique binding specificity for one or more oxidation-specific epitopeson oxidized blood lipoproteins. The E0 antibodies can be used in any ofa number of convenient immunoassay formats (e.g., ELISA, sandwich, etc.)to detect oxidized phospholipids in the methods described herein.

It will be appreciated that the foregoing methods ofdetecting/quantifying oxidized lipids are intended to be illustrativeand not limiting. Numerous other methods of assaying oxidized lipids areknown to those of skill in the art and are within the purview of thisapplication.

D) Contacting the Lipid With an Oxidizing Agent.

In the “second” assay format described above, a lipid or collection oflipids (isolated or present in an LDL) are contacted with an oxidizingagent and the HDL is assayed for the ability to protect the lipids fromoxidization. Essentially any agent capable of oxidizing a phospholipidis suitable for use in this invention. Such agents include, but are notlimited to various peroxides, and in particularly preferred embodimentsthe oxidizing agent is a hydrogen peroxide, 13(s)-HPODE, 15(S)-HPETE,HPODE (hydroperoxyoctadecadienoic acid), HPETE(hydroperoxyeicosatetraenoic acid), HODE, HETE, and the like.

The suitability of other oxidizing agents can be readily determined.This is easily accomplished by contacting an LDL and/or the isolatedphospholipid(s) of interest with the oxidizing agent and measuring theamount of oxidized lipid produced. Alternatively, the “oxidizedlipid/LDL can be assayed for its ability to induce a responsecharacteristic of atherosclerotic plaque formation (e.g., induction ofmonocyte adhesion and/or chemotaxis, and/or differentiation in a cultureof vascular endothelial cells).

E) Scoring the Assay.

The assays are scored as positive for “Group I” HDL (negative for “GroupII” HDL) where the HDL reduces the amount of oxidized lipid or preventslipid from being oxidized in the assay. Conversely, the assays arescored as negative for “Group I” HDL (positive for “Group II” HDL) wherethe HDL does not reduce the amount of oxidized lipid or fails to preventlipid from being oxidized in the assay.

While initial studies indicate that some HDLs offer protection andothers do not, it is neither expected nor required that, on a populationlevel, the distribution of responses be bi-modal. To the contrary, it isexpected that the degree of protection against lipid oxidation or repairof oxidized lipids by HDL will vary with parameters such as genetics,sex, age. physiological maturity, ethnicity, (gestational stage forfemales), general health, immunocompetency of the subject, and the like.

To facilitate the use of a commercial embodiment of the assays for thisinvention, the effects of these (and other) parameters on the protectionafforded by HDL can be routinely determined. Thus, for example, HDLprotection can be assayed in elderly individuals that are diagnosed byother means as very low risk for atherosclerosis and in elderlyindividuals determined to have advanced atherosclerosis. This willprovide a measure of the activity of “protective HDL” or lack ofactivity in “non-protective HDL” among the elderly and permitcomparisons of HDL activity with the young. The effects of these otherparameters can similarly be determined and from such studies populationbaseline “activity” levels for protective and non-protective HDL can bedetermined.

It is emphasized that such measurements need not produce an “absolute”scale to be of considerable use. An evaluation of relative risk is ofgreat use. Because an indication of “elevated” risk for atherosclerosiscan be addressed with relatively little investment (e.g., increasedexercise, dietary changes, increased monitoring, etc.) the downside riskof a false positive (i.e. an indication that the individual is atgreater risk of atherosclerosis) is minor. Similarly, with the presenceof other diagnostic/risk factors for atherosclerosis (e.g., HDL:LDLratios, blood pressure monitoring, behavioral and general healthfactors, etc.) the downside risk of a false negative (i.e. an indicationthat the individual's HDL offers protection against lipid oxidation) isalso relatively slight.

The assay may be scored as positive, negative, or assigned a score on acontinuum (e.g., a particular risk level ranging from very low risk tolow risk to moderate risk to high risk to very high risk, etc.) bycomparison or the assay result to levels determined for the relevantpopulation (e.g., corrected for the various parameters described above)and/or by direct reference to a positive or negative control. Thus, forexample, the results of an assay for change in oxidized lipid caused bycontacting the lipid(s) with the subject's HDL may be compared to a“control” assay run without the HDL (or with the HDL at lowerconcentration). In this instance, a decrease in oxidized lipid in thepresence of the HDL as compared to the assay in the absence of HDLindicates the HDL offers protection/repair of oxidized lipids (i.e., ispositive for “Group I” HDL).

Similarly in an assay where HDL is assayed for the ability to protectlipids from an oxidizing agent, the assay results may be compared with acontrol assay that is identical but lacking the HDL (or having he HDLpresent at lower concentration). Where the assay shows more oxidizedlipid in the absence or reduced HDL, the assay is scored as positive forGroup I HDL.

The assays are scored as positive, as described above, where thedifference between the test assay and the control assay is detectable,and more preferably where the difference is statistically significant(e.g., at least at the 85%, preferably at least at the 90%, morepreferably at least at the 95%, and most preferably at least at the 98%or 99% confidence level).

II. Assay Formats.

The assays of this invention can be practiced in almost a limitlessvariety of formats depending on the particular needs at hand. Suchformats include, but are not limited to traditional “wet chemistry”(e.g., as might be performed in a research laboratory), high-throughputassays formats (e.g., as might be performed in a pathology or otherclinical laboratory), and “test strip” formats, (e.g., as might beperformed at home or in a doctor's office).

A) Traditional Wet Chemistry.

The assays of this invention can be performed using traditional “wetchemistry” approaches. Basically this involves performing the assays asthey would be performed in a research laboratory. Typically the assaysare run in a fluid phase (e.g., in a buffer with appropriate reagents(e.g., lipids, oxidized lipids, oxidizing agent, etc.) added to thereaction mixture as necessary. The oxidized lipid concentrations areassayed using standard procedures and instruments, e.g., as described inthe examples.

B) High-throughput Assay Formats.

Where population studies are being performed, and/or inclinical/commercial laboratories where tens, hundreds or even thousandsof samples are being processed (sometimes in a single day) it is oftenpreferably to perform the assays using high-throughput formats. Highthroughput assay modalities are highly instrumented assays that minimizehuman intervention in sample processing, running of the assay, acquiringassay data, and (often) analyzing results. In preferred embodiments,high throughput systems are designed as continuous “flow-through”systems, and/or as highly parallel systems.

Flow through systems typically provide a continuous fluid path withvarious reagents/operations localized at different locations along thepath. Thus, for example a blood sample may be applied to a samplereceiving area where it is mixed with a buffer, the path may then leadto a cell sorter that removes large particulate matter (e.g., cells),the resulting fluid may then flow past various reagents (e.g., where thereagents are added at “input stations” or are simply affixed to the wallof the channel through which the fluid flows. Thus, for example, thesample may be sequentially combined with a lipid (e.g., provided as anLDL), then an oxidation agent, an agent for detecting oxidation, and adetector where a signal (e.g., a colorimetric or fluorescent signal) isread providing a measurement of oxidized lipid.

In highly parallel high throughput systems samples are typicallyprocessed in microtiter plate formats (e.g., 96 well plates, 1536 wellplates, etc.) with computer-controlled robotics regulating sampleprocessing reagent handling and data acquisition. In such assays, thevarious reagents may all be provided in solution. Alternatively some orall of the reagents (e.g., oxidized lipids, indicators, oxidizingagents, etc.) may be provided affixed to the walls of the microtiterplates.

High throughput screening systems that can be readily adapted to theassays of this invention are commercially available (see, e.g., ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass., etc.). These systems typically automate entire proceduresincluding all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols the various high throughput. Thus, for example, Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

C) “Test Strip” Assay Formats.

In certain preferred embodiments, the assays of this invention areprovided in “test well” or “test strip” formats. In “test well” or “teststrip” formats, the biological sample is typically placed in the well orapplied to a receiving zone on the strip and then a fluorescent orcalorimetric indicator appears which, in this case, provides a measureof the protection or repair afforded by the subject's HDL or componentsthereof.

Many patents have been issued which describe the various physicalarrangements for blood testing. These include systems which involvelateral or horizontal movement of the blood, as well as plasma testing.For example, U.S. Pat. Nos. 4,876,067, 4,861,712, 4,839,297, and4,786,603 describe test carriers and methods for analyticaldetermination of components of bodily fluids, including separatingplasma from blood using glass fibers and the like. These patents, allteach systems which require some type of rotation of test pads or aportion of the test pads during use. U.S. Pat. No. 4,816,224 describes adevice for separating plasma or serum from whole blood and analyzing theserum using a glass fiber layer having specific dimensions andabsorption to separate out the plasma from the whole blood forsubsequent reaction. Similarly, U.S. Pat. No. 4,857,453 describes adevice for performing an assay using capillary action and a test stripcontaining sealed liquid reagents including visible indicators. U.S.Pat. No. 4,906,439 describes a diagnostic device for efficiently andaccurately analyzing a sample of bodily fluid using fluid delivery in alateral movement via flow through channels or grooves.

In addition to the above patents which are representative of the priorart showing various physical types of systems for blood testing and thelike, recent patents have issued which are directed to the particularchemistry for the determination of HDL cholesterol. Thus, U.S. Pat. Nos.4,851,335 and 4,892,815 also to Kerscher et al, describe specific typesof processes and reagents for HDL cholesterol determination.

U.S. Pat. No. 5,135,716 describes a device for determining HDLcholesterol by obtaining plasma from whole blood and determining the HDLcholesterol esterol level from the plasma.

In certain embodiments this invention contemplates devices wherein thesample processing, including plasma separation, HDL metering (ifdesired), contact with a lipid (oxidized or not oxidized), optionalcontact with an oxidizing agent, and detection of oxidized lipids arebuilt into a strip such that user manipulations are minimized and HDLprotective activity can be measured in one to two minutes directly fromwhole blood and/or serum. In a preferred embodiment, the method measuresthe end-point of the reaction and therefore precise time and temperaturecontrols are not necessary.

In certain preferred embodiments the device is similar to that describedin the U.S. Pat. No. 5,135,716. Thus, for example, in one embodiment(see, e.g., FIG. 1), the device includes an inert or active substratesupport 1. A receiving area/receiving reservoir 11 and/or a filteringmembrane, can optionally be present. Disposed in the test device arereagents for the assay typically an oxidized lipid or a lipid that isnot oxidized and an oxidizing agent. The lipid and/or the and anoptional carrier/detection membrane. The test membrane 6 has reactantswhich will react with oxidized lipid and indicate quantify oxidizedlipid, lipid that is not oxidized, or a ratio of oxidized lipid to lipidthat is not oxidized.

In use, blood is added to the blood application area 11 of physicaltransport medium 3. It travels along the channels 2 and physicaltransport medium 3 (e.g., a sheet which is a woven mesh of monofilamentpolyester with 17 micron mesh opening (Tetko, Briarcliff, N.Y.) andhaving a thickness of about 75 microns). Woven fabric, non-woven fabric,gauze and monofilament yarn are among the many choices for the transportmembrane sheet shown as physical transport medium 3. Plasma separationas well as precipitation may be handled by a microporous plasmaseparation membrane 4, in this case, 5 micron nitrocellulose (Schleicherand Schuell, Keene, N.H.).

An optional filtering membrane 5 filters off the LDL and VLDLprecipitates and prevents them from reaching the test membrane 6. Whenpresent, in one embodiment, filtering membrane 5 is a 0.4 micronhydrophilic polycarbonate membrane (Poretics Corp., Livermore, Calif.)used without treatment or 0.2 micron nylon (Micron Separations, Inc.,Westboro, Mass.) or 0.8 micron polysulfone (Gelman Sciences, Ann Arbor,Mich.). The latter two were saturated with 5% or 10% aqueous solution ofpolyethylene glycol (molecular weight 1000 daltons) and dried.Polyethylene glycol (PEG) is optionally used as a wetting agent.

In certain preferred embodiments, test membrane 6, as mentioned,contains enzymes and/or and chromogens and/or fluorescenrs assayingoxidized lipid so that HDL-containing sample reaching it (now devoid ofLDL and VLDL components) reacts with the reagents (e.g., oxidized lipid,lipid that is not oxidized and oxidizing agent(s)) in the test membrane6, producing a colored reaction, the intensity of color beingproportional to oxidized lipid and/or to non-oxidized (reduced) lipid)concentration.

In one preferred embodiment the test membrane 6 is a 0.45 micron nylonmembrane (Micron Separations, Inc, Westboro, Mass.). Top sheet 7 withorifice 12 and transparent area 29 is adhered over the tops of the othercomponents as shown by arrows 8 and 9. Transparent area 29 is comprisedof an aperture covered with a transparent membrane that may or may notbe oxygen permeable.

A drop of blood can be applied to the blood application area 11 ofphysical transport medium 3 through orifice 12 and the calorimetricreaction may be viewed through transparent area 29. Alternatively, oneor more of the layers may be strong enough to support the device in theabsence of an inert substrate support.

One will appreciate that such a laminate device may be designed as atest strip to which a sample is applied, as a “dipstick” for immersioninto a sample, or as a component of a sample receiving receptacle (e.g.,a well in a microtiter plate). It will also be appreciated that thisembodiment is intended to be illustrative and not limiting. Followingthe teaching provided herein and the ample body of literature pertainingto the design of “test strips” such assays for HDL activity according tothe methods of this invention can readily be assembled by those of skillin the art.

V. Kits.

In another embodiment, this invention provides kits for practicing oneor more of the assays described herein. Assay kits preferably compriseone or more containers containing one or more oxidized lipids (isolatedor provided in an LDL), and/or a reduced (not oxidized) lipid and anoxidizing agent (e.g., hydrogen peroxide, 13(S)-HPODE, 15(S)-HPETE,HPODE, HODE, HETE, HPETE, etc.). The kit preferably includes one or morereagents for the detection of oxidized lipids (e.g.,2′,7′-dichlorodihydrofluorescine diacetate, rhodamine, cis-parinaricacid, NBD, cis-parinaric acid cholesteryl ester, diphenylhexatrienepropionic acid, and other fluorescent materials). The kits mayoptionally include any one or more of the devices and/or reagents forpractice of the asssays as described herein. Such devices and/orreagents include, but are not limted to microtiter plates, buffers,filters for quantification of fluorescence, etc.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of theassay methods. Preferred instructional materials describe screening HDL(or components thereof) for the ability to protect lipids fromokidization or to reduce oxidized lipids. The instructional materialsoptionally include a description of the use of such assays forevaluating risk for atherosclerosis.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Normal HDL Inhibits Three Steus in the Formation of MildlyOxidized LDL-Step 1

Apo A-I and an apo A-I peptide mimetic removed “seeding molecules” fromhuman LDL and rendered the LDL resistant to oxidation by human arterywall cells. The apo A-I-associated “seeding molecules” included13-hydroperoxyoctadecadienoic acid [13-HPODE] and15-hydroperoxyeicosatetraenoic acid [15-HPETE]. LDL from micegenetically susceptible to fatty streak lesion formation was highlysusceptible to oxidation by artery wall cells and was rendered resistantto oxidation after incubation with apo A-I in vitro. Injection of apoA-I (but not apo A-II) into mice rendered their LDL resistant tooxidation within 3 hours. Infusion of apo A-I into humans rendered theirLDL resistant to oxidation within 6 hours. HDL and its associated enzymeparaoxonase (PON) also rendered LDL resistant to oxidation. We concludethat: (1) oxidation of LDL by artery wall cells requires “seedingmolecules” that include 13-HPODE and 15-HPETE; (2) LDL from micegenetically susceptible to atherogenesis is more readily oxidized byartery wall cells; (3) Normal HDL and its components can remove orinactivate lipids in freshly isolated LDL that are required foroxidation by human artery wall cells.

Introduction.

HDL and its major apolipoprotein, apo A-I, are known to removecholesterol and phospholipids from cells (Oram and Yokoyama (1996 J.Lipid Res. 37: 2473-2491; Forte et al. (1995). J. Lipid Res. 36:148-157; Bruce et al. (1998) Ann. Rev. Nutr. 18: 297-330; Phillips etal. (1998) Atheroscler. 137 Suppl: S13-S-17). Stocker and colleagues(Christison et al. (1995) J. Lipid Res. 36: 2017-2026) and Fluiter etal. (1999) J. Biol. Chem. 274: 8893-8899, have reported that cholesterylester hydroperoxides can be transferred from LDL to HDL, in part,mediated by cholesteryl ester transfer protein. Fluiter and colleagues(Id.) also demonstrated that there was a selective uptake of oxidizedcholesteryl esters from HDL by rat liver parenchymal cells. Stocker andcolleagues (Garner et al. (1998) J. Biol. Chem. 273: 6080-6087) reportedthat both apo A-I and apo A-II can reduce cholesteryl esterhydroperoxides via a mechanism that involves oxidation of specificmethionine residues (Garner et al. (1998) J. Biol. Chem. 273:6088-6095). However, a direct role for apo A-I in removing oxidizedlipids from lipoproteins and cells has not previously been reported.

Sevanian and colleagues noted that a subpopulation of freshly isolatedLDL that they have described as LDL contains lipid hydroperoxides(Sevanian et al. (1997) J. Lipid Res. 38: 419-428). Parthasarathy(Parthasarathy (1994) Modified Lipoproteins in the Pathogenesis ofAtherosclerosis. Austin, Tex.; R. G. Landes Co. pp. 91-119;Parthasarathy (1994) Free Radicals in the Environment, Medicine andToxicology. edited by H. Nohl, H. Esterbauer, and C. Rice Evans.Richelieu Press, London. pp. 163-179), Witztum and Steinberg (1991) J.Clin. Invest. 88: 1785-1792; Witztum (1994) Lancet 344: 793-795; Chisolm(1991) Clin. Cardiol. 14: 125-130; Thomas and Jackson (1991) J.Pharmacol. Exp. Therap. 256: 1182-1188; Shwaery et al. (1999) Meth. Enz.300: 17-23; Polidori et al. (1998) Free Rad. Biol. Med. 25: 561-567;Thomas et al. (1994) Arch. Biochem. Biophys. 315: 244-254, have studiedLDL oxidation in vitro by metal ions and have hypothesized that LDL mustbe “seeded” with reactive oxygen species before it can be oxidized.Jackson and Parthasarathy suggested a role for lipoxygenases (LO) in the“seeding” of LDL (Parthasarathy (1994) Free Radicals in the Environment,Medicine and Toxicology. edited by H. Nohl, H. Esterbauer, and C. RiceEvans. Richelieu Press, London. pp. 163-179; Thomas and Jackson (1991)J. Pharmacol. Exp. Therap. 256: 1182-1188). They also hypothesized thepossibility that hydrogen peroxide or its lipoperoxide equivalent(Parthasarathy (1994) Free Radicals in the Environment, Medicine andToxicology. edited by H. Nohl, H. Esterbauer, and C. Rice Evans.Richelieu Press, London. pp. 163-179; Thomas and Jackson (1991) J.Pharmacol. Exp. Therap. 256: 1182-1188) may play an important role in“seeding” LDL. We previously reported that de-fatted albumin was capableof removing biologically active lipids from mildly oxidized LDL (Watsonet al. (1995) J. Clin. Invest. 95: 774-782). Based on the known lipidbinding properties of apo A-I (1-4), we reasoned that apo A-I was likelyto be more effective than de-fatted albumin in binding and removinglipids. We, therefore, used apo A-I and apo A-I mimetic peptides totreat LDL. We hypothesized that if apo A-I could bind oxidized lipidsand if the “seeding molecules” were oxidized lipids, then incubating apoA-I and LDL followed by separation of the two, might result in thetransfer of the “seeding molecules” from LDL to apo A-I from which theycould be extracted and identified. We found that both the neutral lipidand fatty acid fractions of the lipids extracted from apo A-I afterincubation with LDL contained “seeding molecules”. The neutral lipidfraction is the fraction where cholesteryl ester hydroperoxides would befound. Since there is evidence that the lipoxygenase pathway can act toform cholesteryl ester hydroperoxides largely as a result of anon-enzymatic process mediated by the products of fatty acid oxidationand alpha tocopherol (Neuzil et al. (1998) Biochem. 37: 9203-9210;Upston et al. (1997) J. Biol. Chem. 272: 30067-30074; Upston et al.(1999) FASEB J. 13: 977-994), we concentrated our efforts on the fattyacid fraction of the lipids extracted from apo A-I after incubation withfreshly isolated LDL. We present evidence in this example that the“seeding molecules” present in freshly isolated LDL are derived, inpart, from the cellular metabolism of linoleic acid(13-hydroperoxyoctadecadienoic acid [13-HPODE]) and arachidonic acid(15-hydroperoxyeicosatetraenoic acid [15-HPETE ]) as originallypredicted by Parthasarathy (10,11) and in accord with the recentfindings of Cyrus et al. that disruption of the 12/15-lipoxygenase genediminished atherosclerosis in apo E-deficient mice (Cyrus et al. (1999)J. Clin. Invest. 103: 1597-1604; Steinberg (1999) J. Clin. Invest. 103:1487-1488).

The experiments presented in this example also indicate that the“seeding molecules” in freshly isolated LDL can be removed and/orinactivated by normal HDL and its components (i.e. apo A-I, andparaoxonase). The experiments detailed in this example and in example 2have led us to propose that the biologically active lipids (Watson etal. (1997) J. Biol. Chem. 272: 13597-13607; Watson et al. (1999) J.Biol. Chem. 274: 24787-24798) in mildly oxidized LDL are formed in aseries of three steps. The first step is the seeding of LDL withproducts of the metabolism of linoleic and arachidonic acid as well aswith cholesteryl hydroperoxides. The second step is trapping of LDL inthe subendothelial space and the accumulation of additional reactiveoxygen species derived from nearby artery wall cells. We propose thatthe third step is the non-enzymatic oxidation of LDL phospholipids thatoccurs when a critical threshold of reactive oxygen species is reachedresulting in the formation of specific oxidized lipids that inducemonocyte binding, chemotaxis, and differentiation into macrophages. Theexperiments in this example focus on the first of these three steps andexample 2 presents data on the second and third steps.

Materials & Methods.

Materials.

Tissue culture materials and other reagents were obtained from sourcespreviously described (Navab, et al. (1991) J. Clin. Invest. 88:2039-2046; Navab et al. (1993). J. Clin. Invest. 91: 1225-1230; Navab etal. (1997) J Clin Invest, 99: 2005-2019). Acetonitrile, chloroform,methanol, ethyl acetate, acetic anyhydride, triethylamine, tert-butanol,polypropylene glycol, ammonium formate, formic acid and water (allOptima grade) were obtained from Fisher Scientific, Pittsburgh, Pa.Authentic L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine(PAPC), and linoleic acid were obtained from Avanti Polar Lipids, Inc.(Alabaster, Ala.). The oxidized phospholipids derived from PAPCincluding Ox-PAPC, and the oxidized phospholipids1-palmitoyl-2-(5)oxovaleryl-sn-glycero-3-phosphocholine (POVPC, m/z594), 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC, nz/z610), and 1-palmitoyl-2-(5,6-epoxyisoprostaneE₂)-sn-glycero-3-phosphocholine (PEIPC, m/z 828) were prepared andisolated as previously described (25,26). 13(S)-HPODE and 15(S)-IPETEwere obtained from Biomol (Plymouth Meeting, Pa.). Human apo A-I and apoA-II and soybean lipoxygenase were obtained from Sigma Chemical Co. (St.Louis, Mo.) and were used for in vitro studies and for injection intomice.

SDS-PAGE analyses demonstrated an approximately 90% purity for apo A-Iand apo A-II preparations. Apo A-I peptide mimetics were synthesized aspreviously described (30-32). Human apo A-I/Phosphatidylcholine discsfor infusion into humans were prepared as previously described by ZLBCentral Laboratory (Bern, Switzerland) (33-35). Purified paraoxonase wasa generous gift from Professor Bert La Du of the University of Michigan.In addition two mutant recombinant paraoxonase preparations, that wereunable to hydrolyze paraoxon (Sorenson et al. (1995) Proc. Natl. Acad.Sci. USA 92: 7187-7191) were utilized.

Lipoproteins.

Low density lipoprotein (LDL, d=1.019 to 1.063 g/ml) and high densitylipoprotein (HDL, d=1.063 to 1.21 g/ml) were isolated based on theprotocol described by Havel and colleagues (Havel et al. (1955) J. Clin.Invest. 43: 1345-1353) from the blood of fasting normal volunteers afterobtaining written consent under a protocol approved by the humanresearch subject protection committee of the University of California,Los Angeles. Lipoprotein deficient serum was prepared by removing thepellet following HDL isolation, dialysis and readjustment of the proteinconcentration to 7.5 g/100 ml. In some experiments butylatedhydroxytoluene (BHT) 20 mM in ethanol was added to freshly isolatedplasma to a concentration of 20 μM and the lipoproteins were separatedby FPLC using methods previously described (Navab et al. (1997) J ClinInvest, 99: 2005-2019). The LDL, HDL and LPDS had endotoxin levels below20 pg/ml which is well below the threshold needed for induction ofmonocyte adhesion or chemotactic activity. The concentration oflipoproteins reported in this study are based on protein content.

Cocultures.

Human aortic endothelial cells (HAEC), and smooth muscle cells (HASMC)were isolated as previously described (Navab, et al. (1991) J. Clin.Invest. 88: 2039-2046). The wells of the microtiter plates were treatedwith 0.1% gelatin at 37° C. overnight. HASMC were added at a confluentdensity of 1×10⁵ cells/cm². Cells were cultured for 2 d at which timethey had covered the entire surface of the well and had produced asubstantial amount of extracellular matrix. HAEC were subsequently addedat 2×10⁵ cells/cm² and were allowed to grow forming a complete monolayerof confluent HAEC in 2 d. In all experiments, HAEC and autologous HASMC(from the same donor) were used at passage levels of four to six.

Monocyte Isolation.

Monocytes were isolated using a modification of the Recalde method aspreviously described (Fogelman et al. (1988) J. Lipid Res. 29:1243-1247) from the blood of normal volunteers after obtaining writtenconsent under a protocol approved by the human research subjectprotection committee of the University of California, Los Angeles.

Monocyte Chemotaxis Assay.

In general, the cocultures were treated with native LDL (250 μg/ml) inthe absence or presence of HDL for 8 h. The supernatants were collectedand used for determination of lipid hydroperoxide levels. The cocultureswere subsequently washed and fresh culture medium 199 (M199) without anyadditions was added and incubated for an additional 8 hrs. This allowedthe collection of monocyte chemotactic activity released by the cells asa result of stimulation by the oxidized LDL. At the end of incubation,the supernatants were collected from cocultures, diluted 40-fold, andassayed for monocyte chemotactic activity. Briefly, the supernatant wasadded to a standard Neuroprobe chamber (NeuroProbe, Cabin John, Md.),with monocytes added to the top. The chamber was incubated for 60 min at37° C. After the incubation, the chamber was disassembled and thenonmigrated monocytes were wiped off. The membrane was then air driedand fixed with 1% glutaraldehyde and stained with 0.1% Crystal Violetdye. The number of migrated monocytes was determined microscopically andexpressed as the mean±SD of 12 standardized high power fields counted inquadruple wells.

Monocyte Adhesion Assay.

In brief, HAEC monolayers, in 48-well tissue culture plates wereincubated with the desired LDL or phospholipid for 4 hrs at 37° C. asdescribed (Watson et al. (1995) J. Clin. Invest. 96: 2882-2891). Afterwashing, a suspension of human peripheral blood monocytes was added andincubated for 10 min. The loosely adherent monocytes were then washedaway, the monolayers were fixed and the number of adherent monocytescounted in 9 high power microscopic fields.

Effect of 13(S)-HPODE on LDL Oxidation.

Freshly isolated LDL (250 μg) was incubated with pure 13(S)-HPODE (1.0μg) in 10% LPDS in M 199 for 4 hrs at 37° C. with gentle mixing. LDL wasre-isolated by centrifugation and was incubated with monolayers of humanaortic endothelial cells. Supernatants were removed at time pointsranging from zero to 5 hours and were assayed for lipid hydroperoxidecontent. The endothelial monolayer was washed after each time point anda monocyte suspension was added, incubated, washed, and the number ofadherent monocytes determined.

Treatment of LDL With Soybean Lipoxygenase.

Freshly isolated LDL (250 μg) was incubated with 10 units of puresoybean lipoxygenase bound to sepharose beads for 4 hrs at 37° C. withgentle mixing. LDL was re-isolated by centrifugation and incubated withmonolayers of HAEC. Supernatants were removed at time points rangingfrom zero to 4 hours and were assayed for lipid hydroperoxide content.The endothelial monolayer was washed after each time point and monocyteadhesion determined.

Mice.

C57BL/6J and C3H/HeJ mice were purchased from Jackson Laboratories (BarHarbor, Me.). All animals were female (4-6 months of age at the time ofthe experiments). The mice were maintained on a chow diet, Purina Chow(Ralston-Purina Co., St. Louis, Mo.) containing 4% fat. LDL was isolatedfrom groups of the lesion susceptible C57BL/6 and from the lesionresistant C3H/HeJ mice from blood obtained from the retroorbital sinususing heparin as an anticoagulant (2.5 U/ml blood) and under mildisoflurane anesthesia, adhering to the regulations set forth by theUniversity of California Animal Research Committee.

Infusion of apo A-I Into Humans.

After obtaining written informed consent and with IRB approval from StBartholomew's and the Royal London School of Medicine and Dentistry, apoA-I/phosphotidylcholine discs were infused at a dose of 40 mg apo A-I/kg of body weight over four hours using the materials and protocoldescribed by Nanjee et al (Nanjee et al. (1999) Arterioscler. Thromb.Vascul. Biol. 19: 979-989; Nanjee et al. (1996) Arteroscler. Thromb.Vascul. Biol. 16: 1203-1214) into six healthy male subjects. The lipidlevels for these six volunters, subjects 1, 2, 3, 4, 5, and 6respectively, were: Total Cholesterol: 149, 160, 164, 209, 153, 163;Triglycerides: 176, 169, 95, 150, 121, 153; LDL-cholesterol: 69, 73, 88,117, 73, 82; and HDL-cholesterol: 45, 54, 56, 62, 59, 47 mg/dl. Plasmawas prepared 2 hrs before and 6 hrs following the infusion, wascryopreserved as described (Havel et al. (1955) J. Clin. Invest. 43:1345-1353) and LDL was isolated by FPLC in the authors' lab in LosAngeles before the experiments. LDL islated from plasma according tothis protocol functions in a manner that is indistinguishable fromfreshly isolated LDL in vitro and in vivo (Rumsey et al. (1994) J. LipidRes. 35: 1592-1598).

Fast Performance Liquid Chromatography (FPLC).

Fast performance liquid chromatography (FPLC) for the rapid and mildisolation of LDL and for re-isolation of LDL and apo A-I afterincubation as shown in FIG. 2 was performed as previously reported(Navab et al. (1997) J Clin Invest, 99: 2005-2019).

Solid Phase Extraction Chromatography.

Solid phase extraction chromatography was preformed as previouslydescribed (Kaluzny et al. (1985) J. Lipid Res. 26: 135-140). In brief,the lipid extract from no more than 2.0 mg LDL protein was resuspendedin 250 μl of chloroform. Solid phase extraction amino columns (Fisher)were conditioned by adding 3.0 ml of methanol followed by 6.0 ml ofhexane using a Vac-Elut manifold (Analytichem International, HarborCity, Calif.). The lipids were added to the column and neutral lipids(cholesterol, cholesteryl esters, cholesteryl ester hydroperoxides,triglycerides, diglycerides, and monoglycerides) were eluted with 3.0 mlof chloroform/isopropanol (2:1, v/v). Free fatty acids were eluted with3.0 ml of 3% acetic acid in diethyl ether, and phospholipids were elutedwith 3 ml of methanol. The solvents were evaporated, the lipids wereresuspended in chloroform/methanol (2:1, v/v with 0.01% BHT, coveredwith argon and stored at −80° C. In these analyses the recovery of theC17:0 added as an internal standard was 92±3%.

Reverse Phase High Performance Liquid Chromatography.

High performance reverse phase liquid chromatography (RP-HPLC) wasconducted according to the methods of Ames and colleagues (Yamamoto andAmes (1987) Free Rad. Biol. Med. 3: 359-361), Kambayashi and colleagues(Kambayashi et al. (1997) J. Biochem. 121: 425-431), and Alex Sevanian(personal communication). In brief, the analyses were performed byinjecting isolated lipids resuspended in mobile solvent onto the columnand eluting with a flow rate of 1.0 ml/min. Detection of fatty acidoxidation products was performed by UV absorbance with a diode arraydetector (Beckman Instruments) scanning from 200 to 350 nm or with anevaporative light scattering detector (SEDEX 55, France). Hypersil MOS-1C8 (Alltech) or Supelcosil LC-18-DB (Supelco) columns were used for theseparation of fatty acid oxidation products, and a Alltima C18 column(Alltech) was used for the separation of cholesteryl ester oxidationproducts. A solvent system composed of methanol/triethylamine(99.99/0.01, v/v) was utilized for eluting 13-HPODE, one consisting of agradient of acetonitrile/water/acetic acid (60/40/0.1, v/v/v) toacetonitrile/water/acetic acid (98/2/0.1, v/v/v) was used for eluting15-HPETE, and one consisting of acetonitrile/2-propanol/water (44/54/2,v/v/v) for eluting cholesteryl linoleate hydroperoxide.

Electrospray Ionization Mass Spectrometry (ESI-MS).

Electrospray ionization mass spectrometry (ESI-MS) in the positive ornegative ion mode was performed according to the protocol and conditionspreviously described (Watson et al. (1997) J. Biol. Chem. 272:13597-13607).

Other Methods.

Protein content of lipoproteins was determined by a modification (Lehmanet al. (1995) In Vitro Cell. Develop.Biol. Animal. 31: 806-810) of theLowry assay (Lowry et al. (1951) J. Biol. Chem. 193: 265-275). Thelevels of monocyte chemotactic protein 1 were determined using an ELISAas described previously (Navab, et al. (1991) J. Clin. Invest. 88:2039-2046). Lipid hydroperoxide levels were measured using the assayreported by Auerbach et al. (1992) Anal. Biochem. 201: 375-380. In someexperiments where indicated the lipids in culture supernatant containingLDL that was oxidized by the artery wall cell cocultures was extractedby chloroform-methanol and hydroperoxides determined by the Auerbachmethod. Paraoxonase (PON) activity was measured as previously described(Gan et al. (1991) Drug Metab. Dispos. 19: 100-106). Statisticalsignificance was determined by model 1 ANOVA. The analyses were carriedout first using ANOVA in an EXCEL application to determine ifdifferences existed among the group means, followed by a pairedStudent's t-test to identify the significantly different means, whenappropriate. Significance is defined as p<0.05.

Results

Apo A-I and An Apo A-I Peptide Mimetic Remove “Seeding Molecules” fromFreshly Isolated Human LDL and Render the LDL Resistant to Oxidation byHuman Artery Wall Cells

Our human artery wall coculture system has been extensivelycharacterized (Navab, et al. (1991) J. Clin. Invest. 88: 2039-2046;Navab et al. (1993). J. Clin. Invest. 91: 1225-1230; Navab et al. (1997)J Clin Invest, 99: 2005-2019; Shih et al. (1996) J. Clin. Invest. 97:1630-1639; Ishikawa et al. (1997) J. Clin. Invest. 100: 1209-1216;Castellani et al. (1997) J. Clin. Invest. 100: 464-474; Shih et al.(1998) Nature 394: 284-287). When LDL is added to this coculture it istrapped in the subendothelial space and is oxidized by the artery wallcells. As a result, three biologically active oxidized phospholipids areproduced—POVPC, PGPC, PEIPC with characteristic m/z ratios of 594, 610,and 828, respectively (Watson et al. (1997) J. Biol. Chem. 272:13597-13607; Watson et al. (1999) J. Biol. Chem. 274: 24787-24798).These three oxidized phospholipids account for the ability of mildlyoxidized LDL to induce endothelial cells to bind monocytes, secrete thepotent monocyte chemoattractant MCP-1, and the differentiation factorM-CSF (Navab, et al. (1991) J. Clin. Invest. 88: 2039-2046; Berliner etal. (1990) J. Clin Invest. 85: 1260-1266; Rajavashisth et al. (1990)Nature 344: 254-257). Conditioned medium from cocultures exposed to LDLwas found to contain MCP-1 (Navab, et al. (1991) J. Clin. Invest. 88:2039-2046). When human monocytes were added to the ILDL-treatedcocultures, the monocytes bound to the endothelial cells and emigratedinto the subendothelial space (Id.). Addition to the cocultures ofneutralizing antibody to MCP-1 completely abolished LDL-induced monocytechemotaxis (Id.). Thus, coculture monocyte chemotaxis is a highlysensitive bioassay for the formation of the biologically active oxidizedphospholipids and the subsequent induction of MCP-1 (Navab, et al.(1991) J. Clin. Invest. 88: 2039-2046; Navab et al. (1993). J. Clin.Invest. 91: 1225-1230; Navab et al. (1997) J Clin Invest, 99: 2005-2019;Ishikawa et al. (1997) J. Clin. Invest. 100: 1209-1216; Berliner et al.(1990) J. Clin Invest. 85: 1260-1266).

Apo A-I is the major protein component of normal HDL. Because of itsknown ability to bind cholesterol and phospholipids (Oram and Yokoyama(1996 J. Lipid Res. 37: 2473-2491; Forte et al. (1995). J. Lipid Res.36: 148-157; Bruceet al. (1998) Ann. Rev. Nutr. 18: 297-330; Phillips etal. (1998) Atheroscler. 137 Suppl: S13-S-17) we hypothesized that apoA-I might also bind the “seeding molecules” in LDL. To test thishypothesis we utilized the protocol shown in FIG. 1. Butylatedhydroxytoluene (BHT) was added to freshly drawn plasma and LDL wasseparated by FPLC and incubated for 2 hours with apo A-I at 37° C. TheLDL and apo A-I were then rapidly separated and studied. We refer to theLDL and apo A-I after separation as “LDL after A-I” and “A-I-after LDL”,respectively.

FIGS. 4A and 4B demonstrate that “LDL after A-I” could not be oxidizedby a coculture of human artery wall cells. The data in FIGS. 3A through3C represent the mean±SD of those obtained in seven out of sevenexperiments using LDL taken from seven different normal individuals andusing different cocultures and monocytes taken from different donors.Thus, these results are highly reproducible and demonstrate in FIG. 4Athat the artery wall cells were unable to oxidize “LDL after A-I”.However, if the lipid extract from “A-I after LDL” was added back to“LDL after A-I”, it was readily oxidized (FIG. 3A). Also, as shown inFIG. 2, “LDL after A-I” did not stimulate monocyte adherence (FIG. 3B)or chemotaxis (FIG. 3C). However, when the lipid extract from “A-I afterLDL” was added back to “LDL after A-I” the reconstituted LDL inducedmonocyte adherence (FIG. 3B) and chemotaxis (FIG. 3C) to the same degreeas sham treated LDL. Results that were highly similar to those shown inFIG. 3C were obtained when monocyte chemotactic protein 1 levels weremeasured by ELISA (data not shown).

The ability of apo A-I to bind lipids has been determined to be afunction of its specific α-helical structure (Palgunachari et al. (1996)Arterioscler. Thromb. Vascul. Biol. 16: 328-338). Anantharamaiah andcolleagues have synthesized apo A-I peptide mimetics that have beenextensively characterized (Garber et al. (1992) Arterioscler. Thromb.12: 886-894; Anantharamaiah (1986) Meth. Enz. 128: 627-647). One ofthese peptide mimetics is known as 37 pA with the amino acid sequenceDWL KAF YDK VAE KLK EAF PDW LKA FYD KVA EKL KEA F (SEQ ID NO: 1). Apeptide with the same amino acid sequence as 37 pA but containing threeextra amino acid residues [aspartic acid (D), glutamic acid (E), andproline (P)] at the N-terminal that prevents the α helix formationnecessary for lipid binding has also been constructed by this groupusing previously published methods (Anantharamaiah (1986) Meth. Enz.128: 627-647). This control peptide, known as 40 P, binds lipids poorlycompared to 37 pA. As shown in FIGS. 4A and 4B, after LDL had beenincubated with and then separated from the apo A-I peptide mimetic 37pA, the LDL (“LDL after 37 pA”) was resistant to oxidation by the arterywall cells (FIG. 4A) and did not induce monocyte chemotactic activity(FIG. 4B). However, if the lipid extract from the peptide afterincubation with the lipoprotein (“37 pA after LDL”) was added back to“LDL after 37 pA”, it was readily oxidized (FIG. 4A). In contrast, “LDLafter 40 P” showed no reduction in LDL oxidation by the artery wallcells (FIG. 4A) and there was no reduction in LDL-induced monocytechemotaxis (FIG. 4B). Thus, both apo A-I and its peptide mimetic 37 pAwere able to remove lipids from freshly isolated LDL that rendered theLDL resistant to oxidation by human artery wall cells and preventedLDL-induced monocyte chemotaxis.

The “Seeding Molecules” in Freshly Isolated LDL that are Removed by ApoA-I Include 13-HPODE and 15-HPETE.

To identify biologically active lipids associated with LDL that wasrapidly isolated by FPLC in the presence of 20 μM BHT as indicated inFIG. 2, lipids were extracted from “A-I after LDL”. These lipids wereseparated by solid phase extraction chromatography. The neutral lipid orfatty acid fractions were then added to cocultures together with eitherPAPC a phospholipid present in LDL or to “LDL after A-I”. Addition tothe cocultures of PAPC or “LDL after A-I” did not stimulate lipidhydroperoxide formation or monocyte chemotactic activity (FIG. 5, panelsA-C, open bars). However, addition to PAPC or to “LDL after A-I” ofeither the fatty acid fraction (FIG. 5, panels A and B, solid bars) orthe neutral lipid fraction (FIG. 5, panels C and D, solid bars)extracted from “A-I after LDL” induced a dose dependent increase in theformation of lipid hydroperoxides and monocyte chemotaxis. Theseexperiments indicated that apo A-I removed lipids from freshly isolatedLDL that was required for the artery wall cells to oxidize both PAPC andLDL. Addition of either the fatty acid or neutral lipid fractionsrecovered from “A-I after LDL” resulted in the oxidation of PAPC and“LDL after A-I” by the artery wall cells.

To further identify the fatty acids, freshly isolated LDL was incubatedwith or without apo A-I and then separated by centrifugation. Followingincubation with apo A-I, the lipids were extracted from the LDL and fromthe apo A-I. Lipids were also extracted from apo A-I that was incubatedwithout LDL (A-I sham) and from LDL that was not incubated with apo A-I(LDL sham). The extracted lipids were then fractionated by reverse phaseHPLC. Apo A-I that had not been incubated with LDL contained little ifany 13-HPODE (FIG. 6, panel A) or 15-HPETE (FIG. 6, panel E). Incontrast, freshly isolated LDL that had been incubated without apo A-I(LDL sham) contained 13-HIPODE and an unidentified nearby peak (FIG. 6,panel B), and also contained 15-HPETE (FIG. 6, panel F). “LDL after A-I”contained substantially less 13-HPODE relative to the nearbyunidentified peak (compare the 13-BIPODE peak relative to theunidentified peak in FIG. 6, panels B and C) and markedly less 15-HPETE(FIG. 6, panel G). FIG. 6, panels D and H, demonstrate that 13-HPODE and15-HPETE, respectively, were transferred to apo A-I. Additional analysesusing mass spectrometry also confirmed the presence of significantamounts of HPODE and HPETE in freshly isolated LDL, both of which wereeffectively removed by incubation with apo A-I (data not shown).Analysis of the lipid extract from “A-I after LDL” by ESI-MS in thenegative ion mode demonstrated the presence of an ion with m/z 311indicating the presence of HPODE (data not shown). An ion present inless abundance compared with that for HPODE and with m/z 335 was alsoobserved, indicating the presence of HIPETE in the lipids of “A-I afterLDL” (data not shown). The lipid extract of “A-I after LDL” alsocontained a relatively large quantity of an ion with nz/z 317 indicatingthe presence of a dehydration product of HPETE i.e. the loss of onemolecule of water (data not shown). Analysis by MS/MS of the lipidextract from freshly isolated LDL that was not treated with apo A-Iconfirmed the presence of HPODE (data not shown). However, HPETE was notdetected in these samples (data not shown). Since HPETE was readilydetected in “A-I after LDL” by both HPLC and MS/MS, we deduce that: 1)treatment of apo A-I may have concentrated the HPETE allowing itsdetection; 2) HPODE may be present in higher concentrations in freshlyisolated LDL than is HPETE.

Addition of authentic 13(S)-HPODE to freshly isolated LDL as describedin Methods increased the formation of lipid hydroperoxides when the LDLwas added to HAEC and also increased monocyte adherence to HAEC (datanot shown).

13(S)-HPODE is the product of lipoxygenase activity on linoleic acid(55,56). Since the major unsaturated fatty acid in LDL is linoleic acid,freshly isolated LDL was incubated with or without soybean lipoxygenase.After incubation with and then separation from the soybean lipoxygenaseas described herein, the LDL was added to HAEC. The LDL that wasincubated with and then separated from soybean lipoxygenasesignificantly increased the formation of lipid hydroperoxides in theculture supernatants and also increased monocyte adherence to HAEC ascompared to LDL incubated without soybean lipoxygenase (data not shown).

Taken together these experiments indicate that the “seeding molecules”in freshly isolated LDL that are removed by apo A-I may include HPODEand HPETE.

Freshly Isolated LDL from Mice that are Genetically Susceptible toAtherosclerosis are Highly Susceptible to Oxidation by Human Artery WallCells and are Rendered Resistant to Oxidation by Human Apo A-I.

When fed an atherogenic diet, C57BL/6J (BL/6) mice develop fatty streaklesions in their aorta while C3H/HeJ (C3H) mice do not, despiteequivalent levels of apo B containing lipoproteins (Paigen et al. (1987)Proc. Natl. Acad. Sci. USA 84: 3763-3767; Ishida et al. (1991) J. LipidRes. 32: 559-568). We previously have presented evidence to suggest thatthe lesion-susceptible BL/6 mice are under oxidative stress Shih et al.(1996) J. Clin. Invest. 97: 1630-1639; Shih et al. (1998) Nature 394:284-287; Liao et al. (1994) J. Clin. Invest. 94: 877-884). A logicalconsequence of this hypothesis might be increased susceptibility tooxidation of LDL from the BL/6 mice compared to LDL from the lesionresistant C3H mice. On a low-fat chow diet the two strains of mice havesimilar low levels of LDL and the lesion susceptible BL/6 mice havehigher levels of HDL (Paigen et al. (1987) Proc. Natl. Acad. Sci. USA84: 3763-3767). To test our hypothesis we incubated freshly isolated LDLfrom the two strains, both of which were on the low-fat chow diet, withand without human apo A-I and then separated the LDL and apo A-I andincubated them with the human artery wall cell cocultures. As shown inFIG. 7, panels A and B, LDL incubated without apo A-I (LDL Sham) fromthe lesion sensitive BL/6 mice was more readily oxidized by the arterywall cells than was the case for the LDL from the lesion resistant C3Hmice (FIG. 7, panel A). In contrast, “LDL after A-I” from both thelesion sensitive BL/6 and the lesion resistant C3H mice were resistantto oxidation by the artery wall cells (FIG. 7, panel A). On the otherhand, if the lipids were extracted from “A-I after LDL”, and added backto “LDL after A-I” the reconstituted LDL was oxidized by the artery wallcells to the same degree as was the case for the sham-treated LDL (FIG.7, panel A). Similar results were obtained for LDL-induced monocytechemotaxis (FIG. 7, panel B). The data in FIG. 7, panels A and B,indicate that the difference in the ability of artery wall cells tooxidize LDL from the lesion sensitive BL/6 mice compared to LDL from theC3H mice is due to lipids in their LDL that can be removed by apo A-I.These data also indicate that this difference is present while theanimals are on the low-fat chow diet.

Injection of Human APo A-I (but not Human Apo A-II) into Mice Rendersthe Mouse LDL Resistant to Oxidation by Human Artery Wall Cells.

To test the ability of apo A-I to alter the potential oxidative state ofLDL in vivo, we injected 100 μg of apo A-I or apo A-II or saline aloneinto mice via their tail veins. Blood was removed immediately (0 hr) or3, 6, or 24 hours after injection. LDL was isolated by FPLC andincubated with human artery wall cocultures and the formation of lipidhydroperoxides and monocyte chemotactic activity was determined. FIG. 8,panel A demonstrates that the freshly isolated LDL from BL/6 mice thathad been injected with apo A-I three to six hours earlier was resistantto oxidation by human artery wall cells and this resistance persistedfor up to 24 hours (FIG. 8, panel A). In contrast, the LDL obtainedimmediately after injection (0 hr) or 6 hours after injection of salinealone, or 6 hours after injection of apo A-II did not render the mouseLDL resistant to oxidation by the artery wall cells (FIG. 8, panel A).Similar results were obtained for monocyte chemotactic activity (FIG. 8,panel B). PON activity in plasma and HDL increased by approximately 20%six hours after injection of apo A-I but did not change after injectionof apo A-II (data not shown). Thus, as was the case for the in vitrostudies above, apo A-I injected in vivo (but not apo A-II) was able todramatically decrease the oxidation of LDL.

Infusion of Human APO A-I into Humans Renders their LDL Resistant toOxidation by Human Artery Wall Cells.

As indicated above in FIG. 8, panel A, injection of apo A-I into micerendered their LDL resistant to oxidation by the artery wall cells. FIG.9, panel A and FIG. 9, panel B, describes a parallel study in humans.Blood was taken from six healthy subjects (one with mildly increasedlevels of triglycerides, 176 mg/dl, as indicated in Methods) two hoursbefore and six hours after infusion of apo A-I. LDL was isolated fromthe plasma at each time point and incubated with human artery wall cellcocultures. As shown in FIG. 9, panel A, in six out of six subjects, theLDL isolated six hours after the infusion of apo A-I was much moreresistant to oxidation by the artery wall cells as compared to the LDLtwo hours before the infusion. Similar results were obtained for LDLinduced monocyte chemotactic activity (FIG. 9, panel B) although thedecrease in oxidation for subject 4 was less than the decrease in LDL-induced monocyte chemotactic activity. PON activity in plasma and HDLwas increased by approximately 20% six hours after the infusion ascompared to two hours before the infusion (data not shown). These dataindicate that as was the case for the mice, injection of apo A-I intohumans rendered their LDL resistant to oxidation by human artery wallcells.

HDL or HDL Associated Enzymes Render LDL Resistant to Oxidation by HumanArtery Wall Cells.

To test the ability of whole HDL and its components other than apo A-I,such as PON, to render LDL resistant to oxidation by artery wall cells,LDL was incubated with or without HDL, or PON, as described herein andthen separated from these and incubated with human artery wall cellcocultures. Incubation with HDL, or PON, rendered the LDL resistant tooxidation by the artery wall cells compared to sham treated LDL (FIG.10, panel A). Similar results were obtained for LDL-induced monocytechemotactic activity (FIG. 10, panel B). Thus, HDL and its associatedenzyme PON can render LDL resistant to oxidation by artery wall cells.

Discussion.

The data presented in this example demonstrate a role for HDL and itscomponents, apo A-I and PON in regulating the first step in a three-stepprocess that leads to the formation of mildly oxidized LDL.Parthasarathy (1994) Modified Lipoproteins in the Pathogenesis ofAtherosclerosis. Austin, Tex.; R.G. Landes Co. pp. 91-119; Parthasarathy(1994) Free Radicals in the Environment, Medicine and Toxicology. editedby H. Nohl, H. Esterbauer, and C. Rice Evans. Richelieu Press, London.pp. 163-179; Witztum and Steinberg (1991) J. Clin. Invest. 88:1785-1792; Witztum (1994) Lancet 344: 793-795; Chisolm (1991) Clin.Cardiol. 14: 125-130; and Thomas and Jackson (1991) J. Pharmacol. Exp.Therap. 256: 1182-1188, hypothesized that LDL must be “seeded” withreactive oxygen species before it can be oxidized. Spector andcolleagues (Spector et al. (1988) Prog. Lipid Res. 27: 271-323;Alexander-North et al. (1994) J. Lipid Res. 35: 1773-1785) havedemonstrated that the lipoxygenase pathway is active in artery wallcells, and Parthasarathy emphasized the possibility that hydrogenperoxide or its lipoperoxide equivalent (Parthasarathy et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1046-1050; Parthasarathy (1994) ModifiedLipoproteins in the Pathogenesis of Atherosclerosis. Austin, Tex.; R.G.Landes Co. pp.91-119; Parthasarathy (1994) Free Radicals in theEnvironment, Medicine and Toxicology. edited by H. Nohl, H. Esterbauer,and C. Rice Evans. Richelieu Press, London. pp. 163-179) may play animportant role in “seeding” LDL. The recent findings of Cyrus et al.(1999) J. Clin. Invest.103: 1597-1604) that disruption of the12/15-lipoxygenase gene diminished atherosclerosis in apoE-deficientmice are consistent with this hypothesis and the data in this example.

We found that freshly isolated LDL from mice on a chow diet that aregenetically susceptible to the development of atherosclerosis was morereadily oxidized by artery wall cells than was the case for LDL takenfrom mice that are genetically resistant to the development ofatherosclerosis. The LDL from both strains of mice was renderedresistant to oxidation by the artery wall cells after apo A-I treatment(FIG. 7, panels A and B), and the levels of oxidation of LDL aftertreatment with apo A-I were not significantly different for the twostrains (FIG. 7, panels A and B). This may indicate that the geneticdifference in susceptibility to develop atherosclerosis may be due, inpart, to a difference in the level of “seeding molecules” in the LDL ofthese two mouse strains.

The in vitro ability of apo A-I (FIG. 3 and FIG. 7) and an apo A-Ipeptide mimetic (FIG. 4, panels A and B) to render LDL resistant tooxidation by artery wall cells was also demonstrated to apply in vivo inboth mice (FIG. 8, panels A and B) and in humans (FIG. 9, panels A andB). In mice, within three hours of injection of apo A-I, LDL wasrendered resistant to oxidation by artery wall cells and this state ofprotection persisted for up to 24 hours (FIG. 8, panels A and B). Incontrast to the case for apo A-I, injection of apo A-II did not protectLDL against oxidation by artery wall cells (FIG. 7, panel A). In humans,infusion of apo A-I into six out of six men rendered their LDL resistantto oxidation by artery wall cells within 6 hours of the infusion (FIG.8, panel A).

Not only was apo A-I capable of favorably altering the susceptibility ofLDL to oxidation by artery wall cells but so was HDL itself and the HDLassociated enzyme, PON. Aviram and colleagues recently demonstrated thatPON has peroxidase activity (Aviram et al. (1998) J. Clin. Invest. 101:1581-1590; Aviram et al. (1998) Arterioscler. Thromb. Vascul. Biol. 18:1617-1624) which in part may explain the role of PON in protectingagainst atherosclerosis in mouse models (Shih et al. (1996) J. Clin.Invest. 97: 1630-1639; Shih et al. (1998) Nature 394: 284-287) and inepidemiological studies (Serrato and Marian (1995J. Clin. Invest. 96:3005-3008; Mackness et al. (1998) Curr. Opin. Lipidol. 9: 319-324;Heinecke and Lusis (1998) Amer. J. Hum. Genet. 62: 20-24). The recentpaper by Dansky and colleagues (Dansky et al. (1999) J. Clin. Invest.104: 31-39) suggested that there was benefit to over expression of apoA-I in apo E deficient mice without an increase in PON activity.However, as acknowledged by the authors of this study (Id.), theylimited their experiments to the first 8 weeks of life. Aviram andcolleagues reported that serum PON activity declined in apo E-deficientmice after 3 months of age, coincident with increases in aortic lesionarea and serum lipid peroxidation (Aviram et al. (1998) J. Clin. Invest.101: 1581-1590). The mice studied by Plump and colleagues (Plump et al.(1994) Proc. Natl. Acad. Sci. USA 91: 9607-9611) were sacrificed at 4 or6 months of age when Aviram's data would suggest that PON activity wouldbe reduced. Dansky and colleagues (Dansky et al. (1999) J. Clin. Invest.104: 31-39) also reported that lipid retention in the artery wall andmonocyte adherence to the endothelium were not different at eight weeksand concluded that the benefit of apo A-I was limited to a later time inlesion development. It should be noted that Dansky and colleagues (Id.)did not measure monocyte adherence but measured instead CD11a adherence,which is not specific for monocytes. Additionally, Dansky and colleagues(Id.) used mice with a genetically mixed background for most of theirexperiments and did not measure monocyte/macrophages in thesubendothelial space. Based on our data, we would predict that apo A-Iover expression might reduce the susceptibility of LDL to oxidationindependent of any change in PON activity. However, we saw approximatelya 20% increase in PON activity six hours after injection of apo A-I (butnot apo A-II) into mice and a similar small increase in humans six hoursafter infusion of apo A-I

Sevanian and colleagues (Sevanian et al. (1997) J. Lipid Res.38:419-428) reported increased levels of cholesterol oxides in LDL. Ourfinding (FIG. 5) that the neutral lipid extracted from “A-I after LDL”could restore the ability of artery wall cells to oxidize “LDL afterA-I” are consistent with Sevanian's observations. Our results on thefatty acid fractions extracted from “A-I after LDL” (FIG. 5, panels A-C,and FIG. 6, panels A-H) indicate that metabolites of the linoleic andarachidonic acids can also act as LDL “seeding molecules”. Review ofFIG. 6, panels A-H reveals that “LDL after A-I” still contained adetectable level of 13-HPODE. However, this level was not sufficient toallow “LDL after A-I” to be oxidized by human artery wall cells (FIGS.3, 5, 7, 8, and 9). Since the step-wise addition of either the neutrallipid or fatty acid fractions from “A-I after LDL” to “LDL after A-I”restored its ability to be oxidized by the artery wall cells (FIG. 5),we conclude that there is a critical threshold for the “seedingmolecules” that is necessary for oxidation.

Stocker and colleagues (Garner et al. (1998) J. Biol. Chem. 273:6080-6087; Garner et al. (1998) J. Biol. Chem. 273: 6088-6095)demonstrated that both apo A-I and apo A-II can reduce cholesteryl esterhydroperoxides via a mechanism that involves oxidation of specificmethionine residues (Garner et al. (1998) J. Biol. Chem. 273:6088-6095). In our experiments only apo A-I and not apo A-II was able toreduce the oxidation of LDL after injection into mice (FIG. 8). Theseresults suggest that the mechanism of protection of apo A-I in ourstudies was different from that investigated by Stocker and colleagues(Garner et al. (1998) J. Biol. Chem. 273: 6080-6087; Garner et al.(1998) J. Biol. Chem. 273: 6088-6095). HDL has been demonstrated to be astrong inverse predictor of risk for atherosclerosis (Miller and Miller(1975) Lancet 1(7897): 16-19). It has been shown to reduceatherosclerosis in animal models when infused (Badimon et al. (1990) J.Clin. Invest. 85:1234-1241) and when associated with the over expressionof apo A-I (Plump er al. (1994) Proc. Nail. Acad. Sci. USA 91:9607-9611). However, the over expression of apo A-II has beendemonstrated to enhance atherosclerosis (Castellani et al. 1997 J. Clin.Invest. 100: 464-474; Warden et al. (1993) Science. 261: 469-472;Hedrick et al. (1993) J. Biol. Chem. 268: 20676-20682). The studiesreported here are consistent with these published reports and indicatethat apo A-I but not apo A-II is capable of removing “seeding” moleculesfrom freshly isolated LDL.

In example 2 we present evidence that normal HDL and its components canalso inhibit the second and third steps in the formation of mildlyoxidized LDL.

Example 2 Normal HDL Inhibits Three Steps in the Formation of MildlyOxidized LDL- Steps 2 and 3

In this example, treatment of human artery wall cells with apo A-I (butnot apo A-II), with an apo A-I peptide mimetic, or with HDL, orparaoxonase, rendered the cells unable to oxidize LDL. Addition of13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramaticallyenhanced the non-enzymatic oxidation of both1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) andcholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE wereapproximately two orders of magnitude greater in potency than hydrogenperoxide in causing the formation of biologically active oxidizedphospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonaseinhibited the biologic activity of these oxidized phospholipids. HDLfrom 10 out of 10 normolipidemic patients with coronary artery disease,who were neither diabetic nor on hypolipidemic medications, failed toinhibit LDL oxidation by artery wall cells and failed to inhibit thebiologic activity of oxidized PAPC while HDL from 10 out of 10 age andsex matched controls did.

We conclude that: (a) Mildly oxidized LDL is formed in three steps, eachof which can be inhibited by normal HDL and, (b) HDL from at least somecoronary artery disease patients with normal blood lipid levels isdefective both in its ability to prevent LDL oxidation by artery wallcells and in its ability to inhibit the biologic activity of oxidizedPAPC.

Introduction.

We discovered that HDL but not apo A-I when added to human artery wallcell cocultures together with LDL prevented the oxidation of the LDL bythe artery wall cells. In those experiments, the apo A-I was kept in theculture together with the artery wall cells and the LDL (Navab et al.(1991) J. Clin. Invest. 88: 2039-2046). Subsequently, in pursuing themechanisms for the ability of HDL to protect LDL against oxidation byhuman artery wall cells, we discovered that if the apo A-I was incubatedwith the cells and then removed prior to the addition of the LDL, theartery wall cells were then unable to oxidize the added LDL. Thissuggested to us that apo A-I might be able to remove from cells not onlycholesterol and phospholipids but perhaps oxidized lipids as well. Thesepreliminary findings prompted us to perform the studies detailed in thisexample.

The experiments detailed in this example and in example 1 have led us topropose that the biologically active lipids in mildly oxidized LDL areformed in a series of three steps. The first step is the seeding of LDLwith products of the metabolism of linoleic and arachidonic acid as wellas with cholesteryl ester hydroperoxides. The evidence for the firststep was presented in example 1. In this example we present evidenceregarding the second step i.e., trapping of LDL in the subendothelialspace and the delivery to this trapped LDL of additional reactive oxygenspecies derived from nearby artery wall cells.

Stocker and colleagues have presented indirect evidence thatlipoxygenases mediate the peroxidation of cholesteryl linoleate largelyby a non-enzymatic process (Neuzil et al. (1998) Biochem. 37: 9203-9210;Upston et al. (1997) J. Biol. Chem. 272: 30067-30074). We demonstrate inthis example that the non-enzymatic oxidation of cholesteryl linoleateis greatly enhanced by the presence of 13-hydroperoxyoctadecadienoicacid [13(S)-HPODE]. We also propose in this example that the third stepin the formation of mildly oxidized LDL is the non-enzymatic oxidationof LDL phospholipids that occurs when a critical threshold of “seedingmolecules” (e.g., 13(S)-HPODE and 15-hydroperoxyeicosatetraenoic acid[15(S)-HPETE] is reached in the LDL. We present evidence in this exampleto indicate that when these “seeding molecules” reach a critical level,they cause the non-enzymatic oxidation of a major LDL phospholipid,1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC). Thisresults in the formation of the three biologically active oxidizedphospholipids: 1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine(POVPC, m/z 594), 1-palmitoyl-2-glutaryl-sn-glycero-3- phosphocholine(PGPC, m/z 610), and 1-palmitoyl-2-(5,6-epoxyisoprostaneE₂)-sn-glycero-3-phosphocholine (PEIPC, m/z 828) (Watson (1999) J. Biol.Chem. 274: 24787-24798; Watson (1997) J. Biol. Chem. 272: 13597-13607).The experiments in this example also indicate that in contrast to thecase for normal HDL, HDL taken from patients with coronary arterydisease who showed normal blood lipid levels, were neither diabetic noron hypolipidemic medications, did not protect LDL against oxidation byhuman artery wall cells and failed to inhibit the biologic activity ofoxidized PAPC.

Materials and Methods.

Materials.

The arachidonic acid analogue, 5,8,11,14-eicosatetraynoic acid (ETYA)was obtained from Biomol (Plymouth Meeting, Pa.). Cholesteryl linoleatehydroperoxide (Ch18:2:—OOH) standard was prepared by peroxidation ofcholesteryl linoleate using tert-butyl hydroperoxide. Seventy percenttert-butyl hydroperoxide was added into the mixture of chloroform andmethanol (2:1, v/v) containing 100 mg of cholesteryl linoleate. Afterperoxidation for 48 hrs at room temperature with mixing, the lipids wereextracted by the Folch method (Folch et al. (1957) J. Biol. Chem. 226:497-509) and separated by reverse phase high performance liquidchromatography (RP-HPLC) as described below. All other materials werefrom sources described in example 1.

Lipoproteins, Cocultures, Monocyte Isolation, Monocyte ChemotaxisAssays, and Monocyte Adhesion Assays.

These were prepared and/or performed as described in example 1.

Patients and Normal Subjects.

Blood samples were collected from patients referred to the cardiaccatheterization laboratory at The Center for Health Sciences at theUniversity of California, Los Angeles. After signing a consent formapproved by the human research subject protection committee of theUniversity of California, Los Angeles, the patient donated a fastingblood sample collected in a heparinized tube. LDL and/or HDL wereisolated by FPLC from the blood samples collected from patients who hadangiographically documented coronary atherosclerosis but who had normaltotal cholesterol (<200 mg/dl), LDL-cholesterol (<130 mg/dl),HDL-cholesterol (males >45 mg/dl, females >50 mg/dl), and triglycerides(<150 mg/dl), who were not on hypolipidemic medications and who were notdiabetic. Data from some patients and some controls previously reportedby us (Navab et al. (1997) J. Clin. Invest. 99: 2005-2019) have beenincluded with additional new data. The inclusion of previously reportedpatients is explicitly indicated in the appropriate figure legend. HDLwas isolated from each individual and paraoxonase activity wasdetermined as previously described (Navab et al. (1997) J. Clin. Invest.99: 2005-2019). The ability of the HDL from each subject to protect LDLagainst oxidation by human artery wall cell cocultures using techniquespreviously described was then determined (Navab et al. (1991) J. Clin.Invest. 88: 2039-2046; Navab et al. (1997) J. Clin. Invest. 99:2005-2019). The LDL used for testing HDL's ability to protect LDLagainst oxidation by human artery wall cells was prepared from a normaldonor and was aliquoted and cryopreserved in sucrose as previouslydescribed (Rumsey et al. (1992) J. Lipid Res. 33: 1551-1561). Todetermine the capacity of HDL to inactivate oxidized phospholipids, insome cases 100 μg/ml of oxidized PAPC (Navab et al. (1997) J. Clin.Invest. 99: 2005-2019) was incubated with 250 μg/ml of HDL in test tubesin 10% LPDS in M199 at 37° C. with gentle mixing. The HDL-Ox-PAPCmixture was then added to endothelial monolayers and monocyte bindingwas determined.

Effect of Over Expression of 15-Lipoxvpenase (15-LO) in Fibroblasts onthe Removal of 13(S)-HPODE by apo A-I.

Fibroblasts that were transfected with vector alone or cells that overexpressed 15-LO were a generous gift of Drs. Joe Witztum and PeterReaven. In the present experiments, the fibroblasts were incubated withor without 100 μg/ml apo A-I. Following 3 hrs of incubation at 37° C.with gentle mixing, the culture supernatants were removed, apo A-I wasseparated by FPLC and the level of hydroperoxides determined in lipidextracts of the culture supernatants and in lipid extracts of apo A-I.

Effect of Lipoxygenase and Cyclooxygenase Inhibitors.

Human artery wall cocultures were preincubated with ETYA at aconcentration of 10⁻⁸ mol/L or withcinnamyl-3,4-dihydroxy-α-cyanocynamate (CDC, from Biomol) at aconcentration of 10⁻⁸ mol/L in M199 containing 10% LPDS for 30 min. Thecocultures were then washed and LDL was added at 250 μg/ml and incubatedfor 8 hrs. The supernatants were removed and assayed for lipidhydroperoxides and monocyte chemotactic activity was determined asdescribed in example 1.

Formation of Oxidized Phospholipids (POVPC, PGPC, and PEIPC) from PAPCBy Addition of 13(S)-HPODE or 15(S)-HPETE or Hydrogen Peroxide.

13(S)-BPODE or 15(S)-HPETE or vehicle alone was added at variousconcentrations to PAPC, mixed and evaporated forming a thin film andallowed to oxidize in air. In some experiments, PAPC was evaporatedforming a thin film and allowed to oxidize in air with 100 μl containinghydrogen peroxide at various concentrations. The samples were extractedwith chloroform/methanol (2:1, v,v) and in the case of the hydrogenperoxide experiments by addition of 5 parts chloroform/methanol(2:1,v,v) to one part aqueous solution, mixing, and centrifugation. Thechloroform phase was collected and analyzed by ESI-MS in the positiveion mode. The level of the remaining PAPC and the oxidized phospholipidsthat formed were determined and expressed in relation to the internalstandard, 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine (DMPC, m/z678.3).

Fast Performance Liquid Chromatography (FPLC) and Reverse Phase HighPerformance Liquid Chromatography (RP-HPLC).

Fast performance liquid chromatography (FPLC) and reverse phase highperformance liquid chromatography (RP-BPLC) were performed as describedin example 1. For the detection of cholesteryl linoleate hydroperoxidean Alltech Alltima 250×4.6 mm, 5 micron RP-HPLC C18 column was used toseparate and detect cholesteryl linoleate hydroperoxide at 234 nm andcholesteryl linoleate at 205 nm. The mobile solvent consisted ofacetonitrile/2-propanol/water (44:54:2, v/v/v) at 1.0 ml/min. Lipidswere resuspended in the mobile solvent for injection.

Electrospray Ionization Mass Spectrometry (ESI-MS).

Electrospray ionization mass spectrometry (ESI-MS) in the positive ornegative ion mode was performed according to the protocol and conditionspreviously described (Watson (1999) J. Biol. Chem. 274: 24787-24798;Watson (1997) J. Biol. Chem. 272: 13597-13607). ESI-MS was performedwith a API III triple-quadrupole biomolecular mass analyzer(Perkin-Elmer) fitted with an articulated, pneumatically assistednebulization probe and an atmospheric pressure ionization source (Watson(1997) J. Biol. Chem. 272: 13597-13607). Positive ion flow injectionanalysis was done with acetonitrile/water/formic acid (50/50/0.1, v/v/v)and negative ion flow injection analysis was done with methanol/water(50/50) containing 10 mM ammonium acetate. For quantitative analysis,1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine (DMPC) orheptadecanoic acid were used as internal standards. Ions were scanned ata step size of 0.3 Da. Data were processed by software provided by PESciex.

Other Methods.

Protein content of lipoproteins was determined by a modification(Lorenzen and Kennedy (1993) Anal. Biochem. 214: 346-348) of the Lowryassay (Lowry et al. (1951) J. Biol. Chem. 193: 265-275). Lipidhydroperoxide levels were measured using the assay described by Auerbachet al. (1992) Anal. Biochem. 201: 375-380. In some experiments, whereindicated, the lipid in culture supernatants containing LDL that wasoxidized by the artery wall cell cocultures was extracted bychloroform-methanol and hydroperoxides determined by the Auerbachmethod. Paraoxonase activity was measured as previously described (Ganet al. (1991) Drug Metab. Dispos. 19: 100-106). Statistical significancewas determined by model 1 ANOVA. The analyses were carried out firstusing ANOVA in an EXCEL application to determine if differences existedamong the group means, followed by a paired Student's t-test to identifythe significantly different means, when appropriate. Significance isdefined as p<0.01.

Results.

Example 1 demonstrated that LDL contains “seeding molecules” necessaryfor LDL oxidation by artery wall cells. We previously reported (Navab etal. (1991) J. Clin. Invest. 88: 2039-2046; Berliner et al. (1990) J.Clin. Invest. 85: 1260-1266) that freshly isolated LDL does not inducemonocyte adherence to endothelial cells and does not induce monocytechemotaxis while mildly oxidized LDL induces both (Id.). The ability ofmildly oxidized LDL to induce monocyte adherence and chemotaxis wasbased on the presence in the mildly oxidized LDL of three oxidizedphospholipids with characteristic m/z ratios (m/z 594,610, and 828)(Watson (1999) J. Biol. Chem. 274: 24787-24798; Watson (1997) J. Biol.Chem. 272: 13597-13607). We did not see evidence of these oxidizedphospholipids in freshly isolated LDL (data not shown). Therefore, weconcluded that the “seeding molecules” in freshly isolated LDL were bythemselves insufficient to generate the three biologically activeoxidized phospholipids either because the level of these “seedingmolecules” was less than some critical threshold or because additionaland different “seeding molecules” were required to generate thebiologically active oxidized phospholipids. Thus, we concluded that atleast one other step in the formation of mildly oxidized LDL wasrequired beyond the initial “seeding”.

Step 2.

Apo A-I (But Not apo A-II) Renders Human Artery Wall Cells Unable toOxidize LDL.

We previously reported that co-incubation of human artery wall cellswith apo A-I and LDL did not protect the LDL against oxidation by theartery wall cells (Navab et al. (1991) J. Clin. Invest. 88: 2039-2046).As shown in FIG. 11, these results were confirmed (compare Co-incubatedA-I to sham treated cultures). However, when the human artery wallcocultures were first incubated with apo A-I and the apo A-I was thenremoved from the cocultures prior to the addition of LDL (Cultures afterA-I), the artery wall cells were not able to oxidize the LDL (FIG. 11,panel A) and monocyte chemotaxis was prevented (FIG. 11, panel B). Incontrast to the case for apo A-I, when the cultures were first incubatedwith apo A-II and the apo A-II then removed, the artery wall coculturesretained their ability to oxidize LDL (FIG. 11, panel A) and inducemonocyte chemotaxis (FIG. 11, panel B) (cultures after A-II).

In other experiments, apo A-I was incubated with a first set ofcocultures and then removed from the first set of cocultures and addedto a second set of cocultures that had been identically treated (i.e.the second set of cocultures had been incubated with apo A-I which wasthen removed). When LDL was added to this second set of cocultures whichcontained apo A-I from the first set of cocultures, these reconstitutedcocultures readily oxidized the LDL (FIG. 11, panel A) and inducedmonocyte chemotaxis (FIG. 11, panel B) (Cultures after A-I+A-I aftercultures).

Similar experiments were performed with apo A-II. Apo A-II was incubatedwith a first set of cocultures and then removed and added to a secondset of cocultures that had been identically treated (i.e. the second setof cocultures had been incubated with apo A-II which was then removed).When LDL was added to this second set of cocultures which contained apoA-II from the first set of cocultures, there was a significant increasein LDL oxidation by the artery wall cells (FIG. 11, panel A) and asignificant increase in LDL-induced monocyte chemotaxis (FIG. 11, panelB) (Cocultures after A-II+A-II after cultures).

Since the reduction in LDL oxidation and LDL-induced monocyte chemotaxisby apo A-I required that the apo A-I be removed from the coculturesafter incubation with the cells and before the addition of LDL (compareCultures after A-I to Co-incubated A-I), we conclude that apo A-Iremoved substances from the artery wall cell cocultures that werenecessary for the LDL to be oxidized by the cocultures and inducemonocyte chemotaxis. We also conclude that apo A-II was incapable ofreducing LDL oxidation and LDL-induced monocyte chemotaxis, and, infact, enhanced these (compare Cultures after A-II to Cultures afterA-I).

Similar results were obtained when the cocultures were treated with anapo A-I peptide mimetic (FIG. 12). The cocultures were incubated with orwithout the apo A-I peptide mimetic 37 pA, and the peptide was thenremoved before the addition of LDL. Other cocultures were incubated withthe control peptide 40 P. Cocultures that had been incubated with theapo A-I peptide mimetic 37 pA that was removed prior to the addition ofLDL were unable to oxidize the added LDL (FIG. 12, panel A) and did notinduce monocyte chemotaxis (FIG. 12, panel B). This was not the casewhen the cocultures were treated with the control peptide 40 P.Following treatment with the control peptide 40 P, LDL was oxidized bythe cocultures (FIG. 12, panel A) and induced monocyte chemotaxis (FIG.12, panel B) to the same degree as sham treated cocultures. We concludethat the apo A-I peptide mimetic 37 pA removed substances from theartery wall cells that were necessary for LDL to be oxidized by thecocultures and induce monocyte chemotaxis.

HDL or HDL Associated Enzymes Render Human Artery Wall Cells Unable toOxidize LDL.

We also tested whether whole HDL and its associated enzyme paraoxonase(PON) could alter the ability of artery wall cells to oxidize LDL. Weincubated the artery wall cell cocultures with HDL, or purified PON andthen removed these prior to the addition of LDL to the cocultures.Treatment of the artery wall cells with any of these two rendered theartery wall cells incapable of oxidizing IDL (FIG. 13, panel A) andprevented LDL-induced monocyte chemotaxis (FIG. 12, panel B). Weconclude that in addition to apo A-I, HDL and PON can prevent humanartery wall cells from oxidizing LDL and inducing monocyte chemotaxis.

Linoleic Acid But Not Oleic Acid Stimulates Human Artery Wall Cells toOxidize LDL.

As noted above, we concluded that the “seeding molecules” in mildlyoxidized LDL were by themselves insufficient to generate the threebiologically active oxidized phospholipids that induce monocytechemotaxis. We hypothesized that this might be because the level ofthese “seeding molecules” was less than some critical threshold orbecause additional and different “seeding molecules” were required togenerate the biologically active oxidized phospholipids in ILDL. Wereasoned that if there was some threshold for the same “seedingmolecules” to generate the oxidized phospholipids and hence monocytechemotaxis and if these “seeding molecules” were in part derived fromthe metabolism of linoleic acid, then enriching the human artery wallcocultures with linoleic acid might be expected to enhance their abilityto oxidize LDL and induce monocyte chemotaxis. Consequently, weincubated human artery wall cocultures with or without linoleic acid(C18:2), or oleic acid (C18:1), washed the cells, and allowed them tometabolize the fatty acids by incubating them for 3 hours at 37° C. infresh medium that was not supplemented with the fatty acids.Subsequently, we tested the ability of these human artery wall cellcocultures to oxidize ILDL and induce monocyte chemotaxis (FIG. 14,panels A-C). Incubating the artery wall cells with linoleic acidsignificantly enhanced the ability of the artery wall cells to oxidizeILDL compared to oleic acid (FIG. 4A) and induce monocyte chemotaxis(FIG. 14, panel B). In other experiments cocultures were incubatedwithout LDL but with (+) or without (−) linoleic acid (C18:2) and thecells were washed and then incubated with or without apo A-I (FIG. 14,panel C). The supernatants were removed and the apo A-I separated byFPLC, and the lipid extracted from the apo A-I. Lipid extracts of theculture supernatants from incubations without apo A-I were alsoobtained. Incubating the cocultures with linoleic acid dramaticallyincreased the 13-HPODE equivalents in the lipid extract of the apo A-I(FIG. 14, panel C) (compare Apo A-I lipid extract of the cells incubatedwith C18:2 to Apo A-I lipid extract of the cells incubated withoutC18:2). We conclude that incubating human artery wall cells withlinoleic acid markedly enhances the cellular production of lipidhydroperoxides, i.e.13-HPODE equivalents which can be removed by apoA-I. We further conclude that incubation of human artery wall cells withlinoleic acid but not oleic acid stimulates the oxidation of LDL byartery wall cells and stimulates LDL-induced monocyte chemotaxis. Inother experiments, the studies described in FIG. 14, panels A-C wereperformed with arachidonic acid. The results indicated that arachidonicacid was even more potent than linoleic acid in stimulating theoxidation of LDL by artery wall cells (data not shown).

Further Evidence for the Role of Lipoxygenase Pathways.

Jackson and Parthasarathy suggested a role for lipoxygenase (LO) in the“seeding” of LDL (Thomas and Jackson (1991) J. Pharmacol. Exp. Therap.256: 1182-1188; Parthasarathy (1994). Modified Lipoproteins in thePathogenesis of Atherosclerosis. Austin, Tex.; R.G. Landes Co. pp.91-119) and Sigari and colleagues demonstrated that fibroblasts overexpressing 15-LO more readily oxidized LDL than fibroblasts transfectedwith vector alone (Sigari et al. (1997) Arterioscler. Thromb. Vascul.Biol. 17: 3639-3645). To further establish the ability of apo A-I toremove lipid hydroperoxide products of the LO pathway from cells, weincubated fibroblasts over expressing LO and cells that were transfectedwith vector alone with apo A-I or without apo A-I as described above.The supernatants were removed, the apo A-I was separated by FPLC, andthe lipid was extracted from the apo A-I. Lipid extracts of the culturesupernatants from incubations without apo A-I were also obtained.Without addition of apo A-I the lipid extracts of the supernatants fromcells over expressing LO contained only slightly more 13-HPODEequivalents compared to the control cells (data not shown). In contrast,the lipid extracts of apo A-I incubated with the cells over expressingLO contained markedly more 13-HPODE equivalents (5.1-fold more) than thelipid extracts of apo A-I incubated with the control cells (data notshown).

Preincubation of the cocultures with the lipoxygenase/cyclooxygenaseinhibitor ETYA (1×10⁻⁸ mol/L) prior to the addition of LDL as describedin Methods resulted in an 80±7% reduction in lipid hydroperoxide levelsand a 75±10% decrease in LDL-induced monocyte chemotactic activity(p<0.008, data not shown). Preincubation of human artery wall cocultureswith the lipoxygenase inhibitor CDC (1×10⁻⁸ mol/L) prior to the additionof LDL as described in Methods resulted in a 73±6% reduction in lipidhydroperoxide levels and a 74±11% decrease in LDL-induced monocytechemotactic activity (p<0.01, data not shown).

Taken together, these experiments suggest that artery wall cells producereactive oxygen species, including those derived from the metabolism oflinoleic and arachidonic acids, that are critical to the oxidation of“seeded” LDL. These experiments also suggest that HDL, apo A-I and PON,can remove or destroy these substances and render the artery wall cellsincapable of oxidizing the “seeded” LDL. Our hypothesis also proposesthat when a critical level in LDL is reached by the further addition ofreactive oxygen species by the artery wall cells to “seeded” LDL, thenon-enzymatic oxidation of a major LDL phospholipid, PAPC, results inthe formation of three biologically active oxidized phospholipids(POVPC, PGPC, and PEIPC) that induce monocyte binding and chemotaxis.

Step 3.

13(S)-HPODE and 15(S)-HPETE Markedly Enhance the Oxidation of PAPC andCholesteryl Linoleate.

We previously reported that if PAPC were exposed to air for 48 hours itwould undergo auto-oxidation to produce the three biologically activephospholipids POVPC, PGPC, and PEIPC) (Watson (1999) J. Biol. Chem. 274:24787-24798; Watson (1997) J. Biol. Chem. 272: 13597-13607). If productsof the lipoxygenase pathway were involved in both the initial “seeding”of circulating LDL and the further “seeding” of LDL by artery wall cellsnecessary to reach a critical threshold that would cause thenon-enzymatic oxidation of PAPC, then the addition of the products ofthe lipoxygenase pathway to PAPC should significantly increase theformation of the three biologically active oxidized phospholipids(POVPC, PGPC, and PEIPC). To test this hypothesis we measured theformation of the three biologically active oxidized phospholipids fromPAPC as a function of time. As shown in FIG. 15, panels A-C the additionof 1.0 μg of 13(S)-HPODE to 10 μg of PAPC enhanced the formation of thethree biologically active oxidized phospholipids at each time pointsampled (POVPC, m/z 594, FIG. 15, panel A; PGPC, m/z 610, FIG. 15, panelB; PEIPC, m/z 828, FIG. 15, panel C).

The data in FIG. 16, panel A, demonstrate that addition of as little as0.5 μg of 13(S)-BPODE to 10 μg of PAPC for 8 hours significantlydecreased the relative abundance of PAPC (m/z 782) and significantlyincreased the formation of the three biologically active oxidizedphospholipids (m/z 594, 610, and 828). FIG. 16, panel B, demonstratesthat addition of as little as 0.5 μg of 15(S)-HPETE to 10 μg of PAPC for8 hours significantly decreased the relative abundance of PAPC (m/z 782)and significantly increased the formation of the three biologicallyactive oxidized phospholipids (m/z 594, 610, and 828).

FIG. 16, panel C, shows that 8 mM hydrogen peroxide added to 10 μg PAPCfor 8 hours dramatically decreased the relative abundance of PAPC andincreased the formation of the biologically active phospholipids, while2 mM and 4 mM hydrogen peroxide had no effect. Since the molecularweight of PAPC is 782 the molar ratio required for the enhancedoxidation of PAPC by hydrogen peroxide was approximately 62:1 (H₂O₂:PAPC) in the experiment described in FIG. 16, panel C. Since themolecular weight of 13(S)-HPODE is 311 and the molecular weight of15(S)-HPETE is 336.5, the molar ratio at which these products of thelipoxygenase pathway promoted the oxidation of PAPC is approximately1:8. Thus, on a molar basis the ability of 13(S)-HPODE and 15(S)-HPETEto oxidize PAPC was more than two orders of magnitude greater than thatof hydrogen peroxide under these conditions. Taken together these dataindicate that 13(S)-HPODE and 15(S)-HPETE, products of linoleic andarachidonic acid metabolism, respectively, act as potent oxidizingagents and promote the non-catalytic oxidation of PAPC to yield thethree biologically active oxidized phospholipids found in mildlyoxidized LDL.

Stocker and colleagues (Neuzil et al. (1998) Biochem. 37: 9203-9210;Upston et al. (1997) J. Biol. Chem. 272: 30067-30074) presented indirectevidence to suggest that the lipoxygenase mediated oxidation ofcholesteryl linoleate is mediated primarily by a non-enzymatic processthat involves products of the lipoxygenase pathway. The experiments inFIG. 17 demonstrate that the presence of 13(S)-HPODE markedly stimulatedthe non-enzymatic formation of cholesteryl linoleate hydroperoxide(Ch18:2-OOH).

Paraoxonase Destroys the Biologic Activity of the Three OxidizedPhospholipids, m/z 594, 610, and 828.

We previously reported that antioxidants and HDL could prevent theformation of biologically active mildly oxidized LDL, but once formedHDL and antioxidants could not decrease the biologic activity of themildly oxidized LDL (Navab et al. (1991) J. Clin. Invest. 88:2039-2046). In these experiments in contrast to those reported abovewhere HDL was incubated with the cocultures before the LDL was added tothe cocultures, we previously had added the HDL together with LDL to thecocultures. In other studies, we reported that PAF-AH (Watson et al.(1995) J. Clin. Invest. 95: 774-782), and PON (Watson et al. (1995) J.Clin. Invest. 96: 2882-2891) could destroy the biologic activity ofmildly oxidized LDL if the enzymes were incubated with the LDL beforeaddition to the cells. These studies were performed with mildly oxidizedLDL, not the specific oxidized phospholipids (i.e. oxidized PAPC orPOVPC, PGPC, PEIPC). To directly test the ability of paraoxonase todestroy the biologic activity of each of the three oxidizedphospholipids we incubated oxidized PAPC (Ox-PAPC), or POVPC, m/z 594;PGPC, m/z 610; or PEIPC, n/z 828 with or without purified paraoxonase asdescribed in Methods. The enzyme was separated from the mixtures and thecompounds were added to human artery wall cocultures. Incubation ofOx-PAPC, or POVPC, m/z 594; PGPC, n/z 610; or PEIPC, m/z 828 withpurified paraoxonase followed by separation of the paraoxonase from thecompounds prior to presentation to the artery wall cell coculturesresulted in the destruction of the biologic activity of each, i.e. theloss of the ability to induce monocyte chemotactic activity (FIG. 18).Two mutant recombinant PON preparations, a generous gift of Drs. RobertSorenson and Bert N. La Du (Sorenson et al. (1995) Proc. Natl. Acad.Sci. USA 92: 7187-7191) were unable to inactivate the biologicallyactive phospholipids in this assay system (data not shown). PON that wasinactivated by boiling at 100° C. had no effect on the activity of theoxidized phospholipids (data not shown).

HDL from Patients with Coronary Artery Disease, With Normal Blood LipidLevels, Who Were Neither Diabetic Nor on Hypolipidemic

Medications, Failed to Prevent LDL Oxidation by Artery Wall Cells andFailed to Destroy the Biologic Activity of Oxidized PAPC.

We reported (Navab et al. (1997) J. Clin. Invest. 99: 2005-2019) thatafter screening more than 250 patients with angiographically documentedcoronary artery disease, we identified 14 patients with angiographicallydocumented coronary artery disease despite normal blood lipid levels andthe absence of diabetes. These 14 had on average lower levels ofparaoxonase activity despite their normal HDL-cholesterol levelscompared to 19 age and sex matched controls (Id.). However, thedifferences between the patient's paraoxonase activity and normalcontrols did not reach statistical significance (Id). We have nowidentified another 10 patients with normal lipid levels (i.e. totalcholesterol <200 mg/dl, LDL-cholesterol <130 mg/dl, HDL-cholesterol >45mg/dl for males and >50 mg/dl for females, and triglycerides <150 mg/dl)who had angiographically documented coronary artery disease, who wereneither diabetic nor on hypolipidemic medications. Combining thepreviously reported data with the new data we now see a statisticallysignificant difference in paraoxonase activity between patients (n=24)and controls (n=29) (FIG. 19, panel A).

Previously we were only able to obtain sufficient sample from 5 of theoriginal 14 patients to test in our coculture system (Navab et al.(1997) J. Clin. Invest. 99: 2005-2019). We reported that HDL from thesefive did not protect against LDL-induced monocyte chemotactic activityin the human artery wall coculture system, while HDL from 4 controlsubjects did. In our current studies we obtained HDL from an additional10 normolipidemic patients with angiographically documented coronaryartery disease, who were neither diabetic nor on hypolipidemicmedications. The ability of HDL from these ten patients and ten age andsex matched normal subjects to modify the oxidation of a control LDL(i.e. LDL obtained from one normal subject which was used in all of theexperiments) is shown in FIG. 19, panel B. As shown in FIG. 19, panel B,HDL taken from 10 out of 10 of the patients did not protect the controlLDL against oxidation by human artery wall cells. Indeed, on average thepatient HDL actually increased control LDL oxidation, while HDL from 10out of 10 age and sex matched normal subjects markedly reduced controlLDL oxidation by the artery wall cells.

Adding the data on monocyte chemotaxis from the ten new patients and tennormal subjects to that of the previously reported 5 patients and fourage and sex matched normal subjects yields a total of 15 patients and 14normal subjects that have now been studied in the coculture system. Inthe experiments shown in FIG. 19, panel C, HDL from 15 out of 15 ofthese patients was unable to protect against LDL- induced monocytechemotactic activity, while 14 out 14 of the controls had HDL which did.

Previously, we had not directly tested the ability of HDL from thissubset of patients to destroy the biologic activity of oxidized PAPC.FIG. 19, panel D demonstrates that the patients (none previouslyreported) had HDL which could not inhibit the biologic activity ofoxidized PAPC (10 out of 10 patients had HDL which did not inhibit thebiologic activity of oxidized PAPC). Indeed, the HDL of the 10 patientson average increased the Ox-PAPC-induced monocyte adherence to HAEC(FIG. 19, panel D). In contrast, HDL from 10 out of 10 age and sexmatched normal subjects markedly decreased the ability of Ox-PAPC toinduce monocyte adherence to HAEC (FIG. 19, panel D). Taken togetherthese data indicate that HDL from this subset of patients with coronaryartery disease appears defective despite their normal plasmaHDL-cholesterol levels.

Discussion.

We have demonstrated in this example that apo A-I and an apo A-I mimeticwere able to act directly on human artery wall cells and profoundlyinfluence their ability to oxidize LDL (FIGS. 11 and 12). In contrast,apo A-II was unable to prevent human artery wall cells from oxidizingLDL (FIG. 11). Similar to the case for LDL (see example 1), treatinghuman artery wall cells with HDL or PON rendered the artery wall cellsincapable of oxidizing LDL (FIG. 13). These experiments indicate thatHDL and its associated enzymes can inhibit human artery wall cells fromcontributing the additional reactive oxygen species necessary forcirculating LDL to reach the critical threshold required to oxidize PAPCto the biologically active phospholipids.

The data in this example support a role for products of the lipoxygenasepathways in artery wall cells in the second step of the formation ofmildly oxidized LDL and are consistent with the recent findings of Cyruset al. that disruption of the 12/15-lipoxygenase gene diminishedatherosclerosis in apoE-deficient mice (Cyrus et al. (1999) J. Clin.Invest. 103: 1597-1604). They concluded that several mechanisms couldexplain their findings but favored one in which “. . .lipoxygenase-derived hydroperoxides or secondary reactive lipid speciesmay be transferred across the cell membrane to ‘seed’ the extracellularLDL, which would then be more susceptible to a variety of mechanismsthat could promote lipid peroxidation.”

The non-enzymatic oxidation of PAPC to form the three biologicallyactive phospholipids (POVPC, PGPC, and PEIPC) was greatly enhanced by13-HPODE and 15-HPETE (FIGS. 15 and 16). Indeed, the ability of 13-HPODEand 15-HPETE to oxidize PAPC to these three biologically activephospholipids was more than two orders of magnitude more potent thanthat of hydrogen peroxide (FIG. 16). These results are consistent withthe findings of Montgomery, Nathan and Cohn (Montgomery et al. (1986)Proc. Natl. Acad. Sci. USA 83: 6631-6635) who found that the amount ofhydrogen peroxide necessary to produce oxidation of LDL was two ordersof magnitude greater than that produced by endothelial cells thatoxidized LDL. The ability of 13-HPODE to stimulate the nonenzymaticformation of cholesteryl linoleate hydroperoxide (Ch18:2-OOH) (FIG. 17′)is consistent with the results of Stocker and colleagues (Neuzil et al.(1998) Biochem. 37: 9203-9210; Upston et al. (1997) J. Biol. Chem. 272:30067-30074) and suggests that products of the lipoxygenase pathway maybe central in the formation of a variety of oxidized lipids ashypothesized by Cyrus et al. (1999) J. Clin. Invest. 103: 1597-1604.

Stocker and colleagues (Garner et al. (1998) J. Biol. Chem. 273:6080-6087; Garner et al. (1998) J. Biol. Chem. 273: 6088-6095) alsodemonstrated that both apo A-I and apo A-II can reduce cholesteryl esterhydroperoxides via a mechanism that involves oxidation of specificmethionine residues (Garner et al. (1998) J. Biol. Chem. 273:6088-6095). In our experiments only apo A-I and not apo A-II was able toreduce the oxidation of LDL after injection into mice (see example 1).Moreover, only apo A-I and not apo A-II was able to decrease the abilityof human artery wall cells to oxidize LDL (FIG. 11).

The destruction of the biologic activity of Ox-PAPC and its components(POVPC, PGPC, and PEIPC) by PON (FIG. 18) and by normal HDL but not byHDL from patients with angiographically proven atherosclerosis despitenormal plasma HDL-cholesterol levels (FIG. 19), suggests that anabnormality in HDL may be responsible, at least in part, for theatherosclerosis in this relatively rare subset of patients. A role forPON in the pathogenesis of atherosclerosis was first suggested by thework of Mackness and Durrington (Mackness et al. (1998) FEBS Let. 423:57-60; Ayub et al. (1999) Arterioscler. Thromb. Vascul. Biol. 19:330-335) and has been supported by the work of a number of laboratoriesincluding ours (Shih et al. (1996) J. Clin. Invest. 97: 1630-1639; Shihet al. (1998) Nature 394: 284-287; Castellani et al. (1997) J. Clin.Invest. 100: 464-474). We report in this example that normolipidemicpatients with coronary artery disease who were neither diabetic nor onhypolipidemic medications had significantly lower levels of PON activitycompared to age and sex matched normal subjects (FIG. 19, panel A).However, there was overlap in PON activities of the patients and normalsubjects. In contrast, HDL from 10 out of 10 patients failed to protectcontrol LDL against oxidation by human artery wall cells (FIG. 19, panelB) and failed to inhibit the biologic activity of oxidized PAPC (FIG.19, panel D) while HDL from 10 out of 10 age and sex matched normalsubjects did. These findings suggest to us that the difference inpatient and control HDL can not be completely explained by differencesin PON activity.

The data presented in this example and in example 1 and in example 1demonstrate a role for HDL and its components, apo A-I, and PON inregulating each and every step in a three step process that leads to theformation of mildly oxidized LDL and which is diagrammed in FIG. 20.Understanding the mechanisms for the formation of mildly oxidized LDLand the role of HDL and its components in preventing the formation andinhibiting the biologic activity of mildly oxidized LDL may lead to newtherapeutic strategies for the prevention and treatment ofatherosclerosis and the clinical syndromes that result from thisinflammatory process.

Example 3

Cell-free Assay to Determine Inflammatory Properties of HDL

The inflammatory properties of HDL play a significant role in thedevelopment of atherosclerosis. We developed a new cell free assay (CFA)to distinguish the inflammatory properties of HDL. This new assay is amodified version of a CFA we previously reported (Navab et al. 2001; JLipid Res. 42:1308-1317) based on the ability of HDL to inactivateoxidized phospholipids in LDL.

HDL was isolated from plasma or serum by Magnetic Bead Reagent(Polymedco). A standard mixture of LDL (90.0 μg/mL) and oxidized PAPC(62.5 μg/mL) were incubated (30 min) and the oxidation of the mixturewas measured in the presence of DCFH-DA (dichlorofluorescein diacetate)that generates a fluorescent signal (485/530 nm) when oxidized. HDL wastested for its ability to inhibit the fluorescent signal, which in turnis a measure of HDL's ability to inactivate oxidized phospholipids inLDL.

Using this new method, we were able to accurately distinguish theinflammatory properties of HDL, i) from mouse models of atherosclerosis,ii) from animal models of atherosclerosis in which an anti-atherogenicpeptide, D-4F (an ApoAl mimetic peptide) was administered, iii) frommonkeys that were administered D-4F, and iv) from known proinflammatoryand anti-inflammatory human samples (see, e.g., FIGS. 21, 22, and 23).Moreover, the new CFA was equally effective on HDL prepared from bothserum and plasma samples (see, e.g., FIG. 24).

HDL function has become the target for therapeutic interventions ofcardiovascular diseases. We have developed a simple, rapid, versatile,and effective CFA to determine the inflammatory properties of HDL inserum samples, which can be used as a biomarker for drug efficacy.

Example 4 Protocols for a Cell-Free Assay

Materials:

Table 1 lists sources for basic materials for a cell-free assayaccording to the present invention.

Table 1. Reagents for use in one embodiment of a cell-free assay for“protective” HDL.

Catalog Materials Vendor Number 1 DCF (2′,7′-dichlorodihydro- MolecularD - 399 fluorescein diacetate, H2DCFDA probes 2 Low-density-lipoprotein(LDL) Biomedical BT - 903 Tech. 3 Methanol (Drisolv) EM Science MX0472-6 4 10× Dulbecco's phosphate Gibco 14200 - 075 buffered saline (PBS) 5Sodium azide Sigma S - 8032 6 Screw cap glass tubes Fisher 7 Corningpolypropylene, round Fisher 07 - 200 - 762 bottom, black microplate 8Vortex mixer Fisher 9 Rotator/nutator 10 Fluorescence plate readerMolecular (e.g., Spectra Max Gemini XS Devices 11 Uniplate (96 wells, 2mL, Whatman 7701 - 5200 polypropylene, round bottom) 12 PAPC 13 HDLMagnetic Bead Reagent Polymedco 5030 14 Cholesterol Assay using ThermoDNA cholesterol standard and cholesterol reagentsMethods:

Preparation of PBS Buffer 1×

Dilute the PBS 10× to 1× using double distilled water. Then add 0.2 g/Lsodium azide. Adjust the pH to 7.4. Filter the solution (0.22 μ filter)and aliquot into, e.g., 50 ml centrifuge tubes. This buffer can bestored at 4° C.

Preparation of Stock Solution of DCF

Prepare a stock solution of 2.0 mg/mL DCF in methanol. Vortex for 2minutes until it completely dissolves. Keep at room temperature for 20minutes. Prepare aliquots of 500 μL in polypropylene tubes. Briefly blowargon gas onto the liquid to prevent further oxidation. Wrap parafilmaround the lid and store at −80° C.

Preparation of Ox-PAPC

Add 5 mg of PAPC (for 100 mg PAPC, 20 glass tubes). Dry down the PAPC inchloroform with argon using a Pasteur glass pipette. (Note: PAPC shouldbe generously spread along the walls of the tubes). Put tubes in hoodfor 48 hrs to air oxidize. After air exposure, add 0.5 ml CHCl₃ to tubeand vortex vigorously. Repeat step 4 for all tubes. Pool the CHCl₃ fromall tubes. Analyze by mass spectrometry to confirm oxidation. Make astock solution of 10 mg/ml Ox-PAPC in Chloroform, aliquot and store at−80° C. protected from light.

Preparation of Stock Solution of Ox-PAPC

From the stock solution of Ox-PAPC (10 mg/ml Ox-PAPC in Chloroform),take appropriate amount needed for the experiment in a glass tube anddry down using Argon gas. Vortex for 5 sec. Resuspend in 1XPBS justbefore use.

HDL Isolation with Magnetic Bead Reagent

Separate plasma or serum from a blood sample using green top tube orserum separator tube by centrifuging at 5° C. at 2300 rpm for 20minutes. Remove the supernatant (serum or plasma). Add ⅕by volume of themagnetic bead reagent to each sample and mix by vortexing. Centrifuge at5° C. at 12,000 rpm for 10 minutes. Remove supernatant (HDL). Using thecholesterol assay (see materials), measure the amount of HDL and preparea stock at a concentration of 200 μg/ml.

LDL+ox-PAPC Pre-incubation.

Prepare LDL at 180 μg/mL using PBS. Prepare Ox-PAPC at 125 μg/mL usingPBS. Make a 1:1 solution of LDL and Ox-PAPC. The final concentrationshould be 90 μg of LDL+62.5 μg of Ox-PAPC/ml. Incubate the mixture for30 minutes at 37° C.

HDL+LDL+ox-PAPC Pre-incubation.

Prepare two sets of HDL serial dilutions. For each set take 20 μg (100μL) of HDL and make serial dilutions (10 μg, 5 μg, and 2.5 μg) to afinal volume of 50 μl in each tube. Add 100 μL PBS to all tubes. The newvolume in all tubes should be 150 μL. To each tube in the first set add100 μL PBS buffer. This will be the “HDL alone” treatment. To each tubein the second set add 100 μL of the LDL+Ox-PAPC mixture. This will bethe “HDL+LDL+ox-PAPC” treatment. In a separate tube, add 150 μL PBS to100 μL of the “LDL+ox-PAPC” mixture. (Note: the final volume in eachtube should now be 250 μL). Incubate both sets for 30 minutes at 37° C.

Re-isolation of HDL

Re-isolate HDL as described above under the “HDL Isolation with MagneticBead Reagent” section. Add DCF to all tubes following Table 2. Incubateat 37° C. for 30 minutes. Read in a plate reader at an excitation of485, emission of 530, and cutoff of 515 nm.

TABLE 2 Allocation of DCF and other components to assay tubes. LDL + Ox-Samples PBS HDL PAPC DCF Total DCF alone 450 50 500 LDL + ox-PAPC alone350 100 50 500 HDL (10 μg) alone 200 250 50 500 HDL (5 μg) alone 200 25050 500 HDL (2.5 μg) alone 200 250 50 500 Reisolated HDL (10 μg) 200 25050 500 Reisolated HDL (5 μg) 200 250 50 500 Reisolated HDL (2.5 μg) 200250 50 500

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of evaluating the risk for atherosclerosis in a mammal, said method comprising: providing a biological sample from said mammal comprising a high-density lipoprotein (HDL); contacting the high-density lipoprotein with an oxidized phospholipid combined with a low density lipoprotein (LDL); and measuring a change in the amount of oxidized or non-oxidized phospholipid wherein the absence of a statistically significant change in the amount of oxidized phospholipid indicates the mammal is at risk for atherosclerosis.
 2. The method of claim 1, wherein said oxidized phospholipid is an oxidized phospholipid that causes a monocytic reaction.
 3. The method of claim 2, wherein said phospholipid is an oxidized form of a lipid selected from the group consisting of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC), 1- stearoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (SGPC), 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (SEIPC), 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (Ox-SAPE), 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylethanolamine (SOVPE), 1-stearoyl-2-glutaroyl-sn-glycero-3-phosphorylethanolamine (SGPE), and 1-stearoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylethanolamine(SEI PE).
 4. The method of claim 1, wherein said oxidized phospholipid is ox-PAPC.
 5. The method of claim 1, wherein said providing comprises removing non-HDL cholesterol from said sample.
 6. The method of claim 5, wherein said providing comprises removing non-HDL cholesterol from said sample using a magnetic bead reagent.
 7. The method of claim 1, wherein said providing comprises isolating the HDL from a blood sample.
 8. The method of claim 1, wherein said measuring comprises a method selected from the group consisting of mass spectrometry, liquid chromatography, thin layer chromatography, fluorimetry, radioisotope detection, antibody detection, and detecting a signal from a label that indicates an oxidized phospholipid.
 9. The method of claim 8, wherein said measuring comprises detecting a signal from a fluorescent indicator.
 10. The method of claim 9, wherein said indicator is selected from the group consisting of 2′,7′-dichlorodihydrofluorescine diacetate, rhodamine, cis-parinaric acid, NBD, cis-parinaric acid cholesteryl ester, dichlorofluorescein diacetate (DCFH-DA), and diphenylhexatriene propionic acid.
 11. The method of claim 9, wherein said indicator is 2′7′-dichlorodihydrofluorescine diacetate (DCFH-DA).
 12. The method of claim 8, wherein said measuring comprises fast performance liquid chromatography (FPLC).
 13. The method of claim 1, wherein said biological sample is whole blood.
 14. The method of claim 1, wherein said biological sample is a blood fraction.
 15. The method of claim 1, wherein said biological sample is serum.
 16. The method of claim 1, wherein said measuring comprises comparing said change in the amount of oxidized phospholipid with the change in amount of oxidized phospholipid produced by contacting the oxidized phospholipid with HDL known to reduce levels of oxidized phospholipid.
 17. The method of claim 1, wherein said measuring comprises comparing said change in the amount of oxidized phospholipid with the change in amount of oxidized phospholipid produced by contacting the oxidized phospholipid with HDL known to be deficient in the ability to reduce levels of oxidized phospholipid.
 18. The method of claim 1, wherein said measuring comprises comparing said change in the amount of oxidized phospholipid with the change in amount of oxidized phospholipid produced by performing the same method without an HDL.
 19. The method of claim 1, wherein said mammal is selected from the group consisting of humans, non-human primates, canines, felines, murines, bovines, equines, porcines, and lagomorphs.
 20. The method of claim 1, wherein said mammal is a human.
 21. The method of claim 1, wherein said mammal is a human diagnosed as having a low HDL:LDL ratio.
 22. The method of claim 1, wherein said mammal is a human diagnosed as being at risk for atherosclerosis by a different method.
 23. The method of claim 1, wherein: said biological sample is serum; and said measuring comprises detecting a fluorescent indicator.
 24. The method of claim 23, wherein said fluorescent indicator is 2′7′-dichlorodihydrofluorescine diacetate DCFH-DA.
 25. The method of claim 24, wherein said oxidized phospholipid is 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC). 